Protein Purification

®
Thermo Scientific Pierce

Protein Purification Technical Handbook
Table of Contents
Thermo Scientific Products for Protein Purification

Introduction 1-2
Binding and Elution Buffers for Affinity Purification 3-5
BupH Buffer Packs 4-5
Solid Supports for Affinity Purification 6-9
Columns for Affinity Purification 8-9
Covalent Coupling of Affinity Ligands to Chromatography Supports 10-25
AminoLink Plus and AminoLink Immobilization Kits and Coupling Resin 14-15
UltraLink Biosupport and Pierce CDI Supports 16
SulfoLink Kits and SulfoLink Coupling Resin 17
UltraLink Iodoacetyl Resin and Micro Peptide Coupling Kit 18
Disulfide Reducing Agents 19
CarboLink Kit, CarboLink Coupling Resin and UltraLink Hydrazide 20-21
PharmaLink Immobilization Kit 22
Pierce Maleic Anhydride Plates and Maleimide Activated Plates 23-24
Avidin:Biotin Binding 26-35
Immobilized Avidin and Streptavidin Products 28
Immobilized NeutrAvidin Products 29
Immobilized Monomeric Avidin and Kit and Immobilized Iminobiotin and Biotin 30
Pierce NeutrAvidin and Streptavidin Coated Plates 32-35
Affinity Purification of Antibodies 36-53
Protein A and Protein G 38-41
Protein A/G and Protein L 42-43
IgG Binding and Elution Buffers for Protein A, G, A/G and L 44-45
Melon Gel Purification Products 46-47
Thiophilic Gel Antibody Purification 48-49
IgM Purification, IgA Purification and Immobilized Jacalin 50-51
Chicken IgY Purification 52
Affinity Purification for Specific Antibodies 53
Immunoprecipitation and Co-Immunoprecipitation Assays 54-62
Pierce Classic and Seize Immunoprecipitation Kits
55-58
Pierce Co-Immunoprecipitation Kits 59
Pierce HA and c-Myc IP/Co-IP Kits 60
Pierce Plus IgG Orientation Kits 61
Affinity Based Contaminant Removal 62-65
Detergent Removing Gel 63
Pierce Blue Albumin Removal Kit 64
Detoxi-Gel Endotoxin Removing Gel 65
Immobilized E. coli Lysate and Kit 65
Additional Affinity Supports 66-75
Immobilized Soybean Trypsin Inhibitor 66
Immobilized Pepstatin 66
Immobilized Heparin 67
Immobilized p-Aminophenyl Phosphoryl Choline 67
Immobilized D-Galactose 67
Immobilized Boronic Acid 67
Pierce Cell Surface Protein Isolation Kit 68-69
Phosphoprotein Enrichment Kit 70
Pierce ConA and WGA Glycoprotein Isolation Kits 71
Pierce Ubiquitin Enrichment Kit 72
™
HisPur Cobalt Resin and Spin Columns 73
MagnaBind Beads and Supports 74
Polystyrene Hydrazide Beads and Polystyrene Beads, Underivatized 75
Affinity Supports 76-79
Appendices 80
Introduction
Activated affinity support products and kits enable a researcher to

immobilize nearly any type of ligand to purify its binding partner(s).
For example, if a peptide antigen is used to immunize animals and
produce antibodies, the same peptide can be immobilized to a
gel support and used to affinity-purify the specific antibody from
animal serum. Alternatively, if a specific antibody is available
against a particular protein of interest, it can be immobilized to
a support and used to affinity-purify the protein from crude cell
lysate. Purification with respect to nearly any binding interaction
can be made by this approach.
Affinity purification products using either immobilized ligands

or activated affinity support chemistries are available for use
in several different formats. Most commonly, porous beaded gel
supports are used for gravity-column, spin-column or batch-scale
purification procedures. Coated microplates are available for
high-throughput screening applications, and magnetic particles
are especially useful for affinity-based cell separation.
Affinity Purification Proteins and other macromolecules of interest can be purified

from crude extracts or other complex mixtures using a variety
Various methods are used to enrich or purify a protein of interest of methods. Precipitation is perhaps the simplest method for
from other proteins and components in a crude cell lysate or separating one type of macromolecule from another. For example,
other sample. The most powerful of these methods is affinity nucleic acids can be precipitated and thereby purified from
purification, also called affinity chromatography, whereby the undesired molecules in solution using ethanol, and proteins can
protein of interest is purified by virtue of its specific binding be selectively precipitated in the presence of ammonium sulfate.
properties to an immobilized ligand. We offer a number of
immobilized protein or ligand products for affinity purification Most purification methods involve some form of chromatography
of antibodies, fusion-tagged proteins, biotinylated proteins and whereby molecules in solution (mobile phase) are separated
other proteins for which an affinity ligand is available. based on differences in chemical or physical interaction with
a stationary material (solid phase). Gel filtration (also called
Affinity purification makes use of specific binding that occurs desalting, size-exclusion chromatography or SEC) uses a porous
between molecules and is used extensively for the isolation of gel material to separate molecules based on size; large molecules
biological molecules. A single pass through an affinity column are excluded from the internal spaces of the gel material while
can achieve a 1,000- to 10,000-fold purification of a ligand from a small molecules enter the resin pores, resulting in a longer path
crude mixture. From a single affinity purification step, it is possible through the column. In ion-exchange chromatography, molecules
to isolate a compound in a form pure enough to obtain a single are separated according to the strength of their overall ionic
band upon SDS-PAGE analysis. interaction with a solid-phase material. By manipulating buffer
conditions, molecules of greater or lesser ionic character can
In affinity purification, a ligand is immobilized to a solid support. be bound to or dissociated from the solid-phase material.
Once immobilized, it specifically binds its partner under mild buffer
conditions (often physiological conditions such as phosphate
buffered saline). After binding to the partner molecule, the support
is washed with additional buffer to remove unbound components
of the sample. An elution buffer is added, disrupting the interaction
between the ligand and its binding partner by pH extremes
(low or high), high salt, detergents, chaotrophic agents or the
removal of some factor required for the pair to bind. Once
released, the binding partner can be recovered from the support
using additional elution buffer. The elution buffer can then be
exchanged by dialysis or desalting into a more suitable buffer
for storage or downstream analysis.
To order, call 800-874-3723 or 815-968-0747. Outside the United States, contact your local branch office or distributor. 1
Introduction
In contrast, affinity chromatography or affinity purification makes groups on the support. However, other coupling approaches are
use of specific binding interactions between molecules. A particular also possible. In the Thermo Scientific GST Orientation Kit (Product
ligand is chemically immobilized or “coupled” to a solid support # 78201), for example, a GST-tagged fusion protein is first bound
so that when a complex mixture is passed over the column, only to an immobilized glutathione support by affinity interaction with
those molecules having specific binding affinity to the ligand are the GST tag and then chemically crosslinked to the support. The
purified. Affinity purification generally involves the following steps: immobilized GST-tagged fusion protein can then be used to affinity-
purify its binding partner(s). Likewise, Thermo Scientific Seize® X
1. Incubate crude sample with the immobilized ligand support Immunoprecipitation Kits (e.g., Product # 45215) and Thermo
material to allow the target molecule in the sample to bind to Scientific IgG Orientation Kits (e.g., Product # 44990) involve
the immobilized ligand. binding and subsequent crosslinking of an antibody to immobilized
2. Wash away unbound sample components from solid support. Protein A or G.
3. Elute (dissociate and recover) the target molecule from the
immobilized ligand by altering the buffer conditions so that Historically, researchers have used affinity purification primarily
the binding interaction no longer occurs. to purify individual molecules of interest. Increasingly, proteomics
research focuses on determination of disease states, cell
differentiation, normal physiological functions and drug discovery
Ligands that bind to general classes of proteins (e.g., Protein A for involving interaction and expression of multiple molecules rather
antibodies) or commonly used fusion protein tags (e.g., glutathione than individual targets. Consequently, the use of affinity methods
for GST-tagged proteins) are available in pre-immobilized forms has expanded to purification of native molecular complexes and
ready to use for affinity purification. Alternatively, more specialized forms the basis for co-immunoprecipitation (co-IP) and “pull-down”
ligands such as specific antibodies or antigens of interest can be assays involving protein:protein interactions.
immobilized using one of several activated affinity supports; for
example, a peptide antigen can be immobilized to a support and There are a variety of activated affinity supports that allow a
used to purify antibodies that recognize the peptide. researcher to purify proteins and other biological molecules of
interest either alone or when present in complexes with their
Most commonly, ligands are immobilized or “coupled” directly to binding partners. Many of these supports are discussed in the
solid support material by formation of covalent chemical bonds following pages.
between particular functional groups on the ligand (e.g., primary
amines, sulfhydryls, carboxylic acids, aldehydes) and reactive
TBS
5ml 5ml 5ml
Antigen 4ml 4ml 4ml
3ml 3ml 3ml
2ml 2ml 2ml

ml
ml
Antigen Antigen
1ml 1ml 1ml
ml
ml Antigen Antigen
ml
1. Immobilize the antigen to 2. Quench the unreacted 3. Add the antibody solution.
an appropriate support. sites and wash.
5ml 5ml 5ml
4ml 4ml 4ml
4. Bind anti-antigen 5. Wash off unbound 6. Elute anti-antigen

antibodies. antibodies. antibodies.
Typical antibody purification using an immobilized antigen column.
2 For more information, or to download product instructions, visit www.thermo.com/pierce

Thermo Scientific Binding and Elution Buffers
for Affinity Purification
Common elution systems for protein affinity purification.
Condition Buffer
pH 100 mM glycine•HCl, pH 2.5–3.0
100 mM citric acid, pH 3.0
50–100 mM triethylamine or triethanolamine, pH 11.5
150 mM ammonium hydroxide, pH 10.5
Ionic strength and/or 3.5–4.0 M magnesium chloride, pH 7.0 in 10 mM Tris
chaotropic effects 5 M lithium chloride in 10 mM phosphate buffer, pH 7.2
2.5 M sodium iodide, pH 7.5
0.2–3.0 sodium thiocyanate
Denaturing 2–6 M guanidine•HCl
2–8 M urea
1% deoxycholate
1 % SDS
Organic 10% dioxane
50% ethylene glycol, pH 8–11.5 (also chaotropic)
Competitor > 0.1 M counter ligand or analog
Binding and Elution Buffers

Most affinity purification procedures involving protein:ligand
interactions use binding buffers, such as phosphate-buffered
saline (PBS), at physiologic pH and ionic strength. This is
especially true when antibody:antigen or native protein:protein
interactions are the basis for the affinity purification. Once the
binding interaction occurs, the support is washed with additional
buffer to remove unbound components of the sample.
Nonspecific (e.g., simple ionic) binding interactions can be

minimized by moderate adjustments to salt concentration or by
adding low levels of detergent in the binding and/or wash buffer.
Finally, elution buffer is added to break the binding interaction and
release the target molecule, which is then collected in its purified
form. Elution buffer can dissociate binding partners by extremes
of pH (low or high), high salt (ionic strength), the use of detergents
or chaotropic agents that denature one or both of the molecules,
removal of a binding factor, or competition with a counter ligand.
In most cases, subsequent dialysis or desalting is required to
exchange the purified protein from elution buffer into a more
suitable buffer for storage or downstream analysis. For more
information on dialysis or desalting, download or request our
a free high-performance dialysis brochure.
The most widely used elution buffer for affinity purification of

proteins is 0.1 M glycine•HCl, pH 2.5–3.0. This buffer effectively
dissociates most protein:protein and antibody:antigen binding
interactions without permanently affecting protein structure.
However, some antibodies and proteins are damaged by low pH,
so eluted protein fraction(s) should be neutralized immediately
by collecting the fractions in tubes containing 1/10th volume of
alkaline buffer such as 1 M Tris•HCl, pH 8.5. Other elution buffers
for affinity purification of proteins are listed in the accompanying
table. In addition, we offer several preformulated binding and
elution buffers designed for affinity purification involving antibodies.
See pages 4–5 and 44–45 for our pre-formulated binding and
elution buffers.
Thermo Scientific Binding and Elution Buffers
for Affinity Purification
BupH™ Dry Buffers BupH Borate Buffer Packs

The most advanced, versatile, time-saving buffer product line available. Ideal for protein modification procedures that require an alkaline pH.
The ultimate in convenience Each pack yields 50 mM borate, pH 8.5 after adding 500 ml of
1. Reach for the sealed foil pack sitting conveniently on deionized water (20 L total).
your bench top.
2. Open and add to deionized water. Ordering Information
3. The fresh buffer is ready to use in practical aliquots so
there’s no waste. Product # Description Pkg. Size
28384 BupH Borate Buffer Packs 40 pack
The ultimate in versatility
• Routine buffers are designed for use in affinity purification
and a variety of other applications. BupH Carbonate-Bicarbonate Buffer Packs
• Using one buffer source maintains consistency and eliminates
variables within the lab. Ideal for microplate coating for RIA and EIA techniques.
• Specialized buffers ideally support your work in specific Each pack yields 0.2 M carbonate-bicarbonate buffer, pH 9.4 when
chemistries and methods. dissolved in 500 ml deionized water (20 L total).
The ultimate in integrity Ordering Information

• Unlike stored buffers, BupH Buffers are protected from
contamination. Product # Description Pkg. Size
• Carry out applications with confidence in buffer quality.
28382 BupH Carbonate-Bicarbonate Buffer Packs 40 pack
• “Test-assured” with our commitment to world-class, quality
management standards.
BupH Citrate-Carbonate Buffer Packs
The ultimate in time savings
• The making of specialized and routine buffers is no longer Great for use with Thermo Scientific UltraLink® Supports.
time-consuming.
• No component measurement, pH adjustment, quality validation, Each pack yields 0.6 M sodium citrate, 0.1 M sodium carbonate, pH
preparation tracking or refrigeration hassles. 9 when dissolved in 100 ml of deionized water (1 L total).
• Move forward with your work by eliminating re-tests due
to buffer problems. Ordering Information
Product # Description Pkg. Size

28388 BupH Citrate-Carbonate Buffer Packs 10 pack

BupH Citrate-MOPS Buffer Packs BupH Phosphate Buffered Saline Packs
Great for use with Thermo Scientific UltraLink Supports. Ideal for crosslinking and biotinylation.
Each pack yields 0.6 M sodium citrate, 0.1 M MOPS buffer, pH 7.5 Each pack yields 500 ml of 0.1 M phosphate, 0.15 M NaCl, pH 7.2
when dissolved in 100 ml of deionized water (1 L total). when dissolved in 500 ml deionized water (20 L total).
Ordering Information Ordering Information
Product # Description Pkg. Size Product # Description Pkg. Size

28386 BupH Citrate-MOPS Buffer Packs 10 pack 28372 BupH Phosphate Buffered Saline Packs 40 pack
BupH MES Buffered Saline Packs BupH Tris Buffered Saline Packs
Ideal for use with carbodiimide-coupling chemistries. Ideal all-purpose binding buffer.
BupH MES Buffered Saline Packs are designed for use with car- Each pack yields 500 ml of 25 mM Tris, 0.15 M NaCl, pH 7.2 when
bodiimide-coupling chemistries, such as CarboxyLink Coupling dissolved in 500 ml deionized water (10 pack makes 5 L total; 40
Resin (Product # 20266) plus EDC. One pack of BupH MES Buffered pack makes 20 L total).
Saline dissolved in 500 ml of water yields 0.1 M MES (2-[N-Morpho
lino]ethanesulfonic acid), 0.9% NaCl, pH 4.7. Ordering Information
Ordering Information Product # Description Pkg. Size

28376 BupH Tris Buffered Saline Packs 40 pack
28379 BupH Tris Buffered Saline Packs 10 pack
28390 BupH MES Buffered Saline Packs 10 pack
BupH Modified Dulbecco’s PBS Packs

A ready-to-use PBS for immunoassays.
Each pack yields 500 ml of 0.008 M sodium phosphate, 0.002 M

potassium phosphate, 0.14 M sodium chloride and 0.01 M
potassium chloride, pH 7.4 when dissolved in 500 ml deionized
water (20 L total).
Ordering Information

28374 BupH Modified Dulbecco’s PBS Packs 40 pack
Solid Supports for Affinity Purification
Porous Beaded Resins

Porous beaded supports generally provide the most useful properties
for affinity purification of proteins. We offer affinity purification
products in two main porous gel support formats: crosslinked
beaded agarose and Thermo Scientific UltraLink Biosupport.
The various features of these two supports are listed in the
accompanying table. Agarose is good for routine applications but
crushes easily, making it suitable for gravity-flow column or small-
scale batch procedures using low-speed centrifugation. UltraLink
Biosupport is incompressible and can be used in high-pressure
applications with a peristaltic pump or other liquid chromatography
system. In addition, UltraLink Supports display extremely low
nonspecific binding characteristics because they are polyacrylamide-
based. Both supports perform well in typical gravity-flow and spin
column purification and immunoprecipitation procedures.
Affinity Purification Supports Magnetic Particles

Affinity purification involves the separation of molecules in solution When a matrix is required for affinity purification of cells within a
(mobile phase) based on differences in binding interaction with a population, we recommend Thermo Scientific MagnaBind™ Beads.
ligand that is immobilized to a stationary material (solid phase). Magnetic affinity separation is a convenient method for isolating
A support or matrix in affinity purification is any material to which antibodies, antigens, lectins, enzymes, nucleic acids and cells
a biospecific ligand may be covalently attached. Typically, the while retaining biological activity. Samples containing the molecule
material to be used as an affinity matrix is insoluble in the system of interest are incubated with beads that are derivatized with an
in which the target molecule is found. Usually, but not always, antibody or other binding partner. A rare earth magnet is used to
the insoluble matrix is a solid. Hundreds of substances have been pull the MagnaBind Beads out of solution and onto a surface.
described and employed as affinity matrices. The buffer can be carefully removed, containing any non-bound
molecules or cells.
Useful affinity supports are those with a high surface area to
volume ratio, chemical groups that are easily modified for covalent MagnaBind Beads consist of a silanized surface over an iron
attachment of ligands, minimal nonspecific binding properties, oxide core (see Table). The silanized surface has been derivatized
good flow characteristics, and mechanical and chemical stability. to contain active groups, such as carboxylic acids or primary
When choosing an affinity support or matrix for any separation, amines, or specific affinity molecules such as streptavidin; Protein
the most important question to answer is whether a reliable A; Protein G; or goat anti-mouse, anti-rabbit or anti-rat IgG. Due to
commercial source exists for the desired matrix material in the the nature of the MagnaBind Beads, strong elution conditions are
quantities required. Fortunately, we offer a wide range of practical not recommended with these products. When using MagnaBind
and efficient matrices in volumes ranging from 1 ml to much larger Beads to purify certain cells from a population, elution procedures
bulk quantities. are not required, as the beads automatically dissociate from the
cells within 48 hours due to cell surface turnover. See page 74
for a complete listing of MagnaBind Supports.

Physical properties of porous gel supports.
4% Agarose 6% Agarose Thermo Scientific UltraLink Biosupport

Support (crosslinked beaded agarose) (crosslinked beaded agarose) (co-polymer of crosslinked bis-acrylamide and azlactone)
Bead 45–165 µm 45–165 µm 50–80 µm
Exclusion Limit 20,000,000 daltons 4,000,000 daltons 2,000,000 daltons (1,000 Å pore size)
Durability Crushes under pressure Crushes under pressure Sturdy (> 100 psi, 6.9 bar)*
Types of Chromatography Gravity and small spin columns Gravity and small spin columns FPLC Systems, medium pressure, gravity flow
Coupling Capacity Medium Medium High
pH range 3–11 3–11 3–11
Form Preswollen Preswollen Dry or Preswollen
* Note: The indicated maximum pressure of 100 psi refers to the maximum pressure
drop across the gel bed that the support can withstand. It does not necessarily refer
Microplates
to the indicated system pressure shown on a liquid chromatography apparatus because
the system pressure may not actually be measuring the pressure drop across the Polystyrene microplates are another type of matrix commonly used
column. Typical system pressures are usually much higher due to pumping through for immobilization of proteins. Proteins passively adsorb to the
small I.D. tubing, auto-samplers, detectors, etc. When packed into a 3 mm ID x 14 cm
height glass column, these exclusive supports have been run to approximately 650 psi polystyrene surface through hydrophobic interactions. Generally,
(system pressure) with no visual compression of the gel or adverse effects on this adsorption of proteins onto the polystyrene surface occurs
chromatography. These columns can be run at linear flow rates or 85–3,000 cm/hour best in carbonate/bicarbonate buffer at an alkaline pH (9.0–9.5).
with excellent separation characteristics.
In addition, polystyrene surfaces can be derivatized with certain
Characteristics of underivatized Thermo Scientific MagnaBind Beads.
chemistries that will allow peptides and other nonprotein molecules
to adhere to the surface to perform affinity assays in the wells of
Composition Silanized iron oxide the plates.
Magnetization 25–35 EMU/g
We offer precoated plates to allow researchers an easy-to-use,
Type of Magnetization Superparamagnetic (no magnetic memory) consistent method for affinity purification or identification of
Surface Area > 100 m2/g specific molecules of interest. The plates offered include those
Settling Rate 4% in 30 minutes
specific for fusion proteins (6xHis, GST and GFP), antibodies
(Protein A, Protein G, Protein A/G, Protein L, goat anti-mouse and
Effective Density 2.5 g/ml
goat anti-rabbit IgG), biotin (streptavidin and Thermo Scientific
Number of Beads 1 x 108 beads/mg NeutrAvidin® Protein) and those with reactive chemistries (maleic
pH Stability Aqueous solution, above pH 4.0 anhydride and maleimide) to allow binding of nonprotein samples
Concentration 5 mg/ml
that do not adsorb to the plastic microplate well surface. Only
selected microplate products are featured in this handbook.
Note: To establish a microbe-free preparation, MagnaBind Beads can be washed with
antibiotic medium or γ-irradiated.
Thermo Scientific Pierce Columns for Affinity Purification
Spin Cups and Columns Spin Columns – Snap Cap

Thermo Scientific Pierce Spin Columns are convenient tools for Snap cap with collection tubes.
manipulating small volumes of affinity supports (5–500 µl) for
protein purification. Add the affinity resin and sample to one of Highlights:
the columns, use a microcentrifuge to efficiently wash away con- • Used in the Cell Surface Protein Isolation
taminants and elute your purified sample without losing any resin and Glycoprotein Isolation Kits
in the process. Spin columns allow you to affinity-purify • Column Volume: 1,000 µl
more protein in less time! • Resin Volume: 20–500 µl
• Filter Type: Polyethylene, ~30 µm pore size
Highlights:
• Cap Type: Snap cap on column (no cap on collection tube);
• Efficient washing of samples means fewer washes are needed
press-on bottom caps
to remove contaminating proteins
• Efficient elution of samples means more antigen and
co-precipitated proteins are recovered
• No resin loss means more consistent IP and co-IP results Micro-Spin Columns
• No need to decant supernatant from IP or co-IP pellet • Column Volume: 400 µl
• Spin protocols drastically reduce the time required for IPs and co-IPs • Resin Volume: 5–100 µl
• Low protein-binding polypropylene column construction • Filter Type: Polyethylene, ~30 µm pore size
minimizes nonspecific binding • Cap Type: O-ring screw top caps; press-on bottom caps
Spin Cups – Paper Filter Ordering Information

Paper filter with collection tubes.
Highlights: 69700 Spin Cups – Paper Filter 50/pkg
Includes cups and collection tubes
• Paper filters are resistant to clogging from
cellular debris 69715 Microcentrifuge Tubes 72/pkg
Collection Tubes for Product # 69700
• Column Volume: 600 µl
• Resin Volume: 20–300 µl 69702 Spin Cups – Cellulose Acetate Filter 50/pkg
Includes cups and collection tubes
• Filter Type: Paper, ~10 µm pore size
69720 Microcentrifuge Tubes 72/pkg
• Cap Type: Collection tube cap fits onto inserted spin cup Collection Tubes for Product # 69702
69705 Spin Columns – Screw Cap Kit

with Luer-Lok Adaptors
Spin Cups – Cellulose Acetate Filter Includes: Spin Columns, Screw Caps and Column Plugs
Luer-Lok Adaptors
25 each
5 each
Large Frits (6.8 mm diameter 10 µm pore size) 25 each
Cellulose acetate filter with collection tubes. Small Frits (2.7 mm diameter 10 µm pore size) 25 each
Large and Small Frit Tools 1 each
Highlights:
69725 Spin Columns – Snap Cap Kit
• Used in our IP and Co-IP Kits with Collection Tubes
• Column Volume: 800 µl Includes: Spin Columns and Bottom Caps 50 each
Collection Tubes 100 each
• Resin Volume: 20–400 µl
89879 Micro-Spin Columns 50/pkg
• Filter Type: Cellulose acetate, 0.45 µm pore size
• Cap Type: Collection tube cap fits onto inserted spin cup
Disposable Plastic Columns

Spin Columns – Screw Cap Automatic “stop-flow” action provided by porous polyethylene
™
Screw cap with Luer-Lok Adaptors. discs prevents column beds from drying out.
Highlights: Highlights:
• Luer-Lok Adaptors allow these columns to be used • Supplied complete with porous polyethylene discs, stoppers and
for syringe-based purifications end caps
• Column Volume: 900 µl • Compatible with most types of aqueous buffer eluents commonly
• Resin Volume: 20–400 µl used in chromatography
• Filter Type: Polyethylene, ~10 µm pore size • Can be pre-packed and stored until needed
• Small & large frit options for different sample sizes
• Cap Type: O-ring screw top caps; press-in bottom plugs

2 ml Centrifuge Columns
Highlights:
Product # Description Pkg. Size • Total Volume: 5 ml (2 ml resin bed, 3 ml reservoir)
29920 Disposable Polystyrene Columns 100/pkg • Resin Volume: 2 ml
Ideal for packing 0.5–2.0 ml bed volumes.
29922 Disposable Polypropylene Columns 100/pkg
Ideal for packing 1–5 ml bed volumes.
• Receiver Tube: Fits standard 15 ml conical
centrifuge tubes
29924 Disposable Polypropylene Columns 100/pkg
Ideal for packing 2–10 ml bed volumes. • Cap Type: Screw-top cap
29923 Disposable Polypropylene Funnels
• Twist-off bottom and press-on cap to reseal
50/pkg
Buffer reservoirs that fit Product #’s 29920,
29922 and 29924. 5ml
29925 Disposable Column Trial Pack Trial Pack 5 ml Centrifuge Columns 3ml
4ml
Includes accessories plus two each of Product #’s 2ml
29920, 29922 and 29924 and one of Product # 29923. Highlights: 1ml
• Total Volume: 8 ml (5 ml resin bed, 3 ml reservoir)

• Resin Volume: 5 ml
Centrifuge Columns
Efficiently handle a wide variety of resin volumes for affinity • Receiver Tube: Fits standard 15 ml conical
purification! Thermo Scientific Pierce Centrifuge Columns are centrifuge tubes
convenient tools for handling 40 µl–10 ml of an affinity purification • Cap Type: Screw-top cap
support. Add the affinity resin to one of the polypropylene columns, • Twist-off bottom and press-on cap to reseal
remove the twist-off bottom and allow the resin to pack itself.
Then add your sample and allow it to bind to the support. Use a 10ml
centrifuge to efficiently wash away any contaminants and elute 9ml
10 ml Centrifuge Columns
8ml
7ml
your purified protein. 6ml

5ml
4ml
3ml
Highlights: 2ml
Pierce Centrifuge Columns allow you to use a spin-column format 1ml
in addition to traditional gravity flow to reduce the time required for • Total Volume: 22 ml (10 ml resin bed, 12 ml reservoir)
column washing and elution. This accelerates sample processing • Resin Volume: 10 ml
time and makes multiple-sample processing possible. Centrifuge • Filter Type: Polyethylene, ~30 µm pore size
columns allow you to affinity-purify more protein in less time! • Receiver Tube: Fits standard 50 ml conical
centrifuge tubes
Centrifuge columns are made from low protein-binding • Cap Type: Screw-top cap
polypropylene for compatibility with protein purification, and
• Twist-off bottom and press-on cap to reseal
they fit into standard centrifuge tubes for use in any centrifuge.
Applications for Centrifuge Columns:

• Affinity purification/affinity chromatography
• Immunodepletion
• Spin desalting
89868 Centrifuge Columns, 0.8 ml capacity Kit
Includes: Pierce Centrifuge Columns 50 each
0.8 ml Centrifuge Columns Screw Caps 50 each
Highlights: 89869 Centrifuge Columns, 0.8 ml capacity Kit

Includes: Pierce Centrifuge Columns 4 x 50 each
• Total Volume: 800 µl Screw Caps 4 x 50 each
• Resin Volume: 40–400 µl 89896 Centrifuge Columns, 2 ml capacity Kit
• Filter Type: Polyethylene, ~30 µm pore size Includes: Pierce Centrifuge Columns 25 each
Screw Caps and Tips 25 each
• Receiver Tube: Fits standard microcentrifuge tubes
(e.g., Product # 69720) 89897 Centrifuge Columns, 5 ml capacity Kit
• Cap Type: O-ring screw-top cap Screw Caps and Tips 25 each
• Twist-off bottom 89898 Centrifuge Columns, 10 ml capacity Kit
Screw Caps and Tips 25 each
69707 Column Extender 10 each

Fits 89896, 89897 and 89898. Increases column
capacity by 35 ml
Covalent Coupling of Affinity Ligands
to Chromatography Supports
The concept of using immobilized affinity ligands to target

biomolecules has extended beyond chromatographic applications.
Affinity ligands are now coupled to latex beads, nanoparticles,
macro-beads, membranes, microplates, array surfaces, dipsticks
and a host of other devices that facilitate the capture of specific
biomolecules. The application of affinity targeting includes
purification, scavenging (or removal of contaminants), catalysis
(or modification of target molecules) and a broad range of
analytical uses to quantify a target molecule in a sample solution.
Designing custom affinity supports that are able to target unique

biomolecules requires methods to covalently link a ligand to an
insoluble matrix. Regardless of the intended application, the
chemical reactions that make ligand attachment possible are
well characterized and facilitate the attachment of biomolecules
through their common chemical groups. The types of functionalities
generally used for attachment include easily reactive components
such as primary amines, sulfhydryls, aldehydes, carboxylic acids,
hydroxyls, phenolic groups and histidinyl residues. Usually, the
Covalent Immobilization of Ligands solid-phase matrix first is activated with a compound that is
reactive toward one or more of these functional groups. The
Affinity chromatography uses the specific interactions between two activated complex then can form a covalent linkage between
molecules for the purification of a target molecule. In practice, a the ligand and the support, resulting in ligand immobilization.
ligand having affinity for a target molecule is covalently attached to
an insoluble support and functions as bait for capturing the target The type of linkage that is formed between the matrix and the
from complex solutions. immobilized ligand affects the performance of the affinity support
in a number of ways. A linkage that allows the coupled ligand to
The affinity ligand can be virtually any molecule that can specifically leach from the matrix will result in contamination of the purified
bind the target without displaying significant nonspecific binding protein and shorten the useful life of the affinity support. A linkage
toward other molecules in the solution. Ligands that have been that introduces a charged functional group into the support can
used for affinity separations include small organic compounds that cause nonspecific binding by promoting ion-exchange effects.
are able to dock into binding sites on proteins, inorganic metals that A linkage that alters the structure of the matrix can change the
form coordination complexes with certain amino acids in proteins, flow and binding characteristics of the support. Cyanogen
hydrophobic molecules that can bind nonpolar pockets in bromide (CNBr)-activated supports are informative as an example
biomolecules, proteins with specific binding regions that are able to of these principles. This popular immobilization method results
interact with other proteins, and antibodies, which can be designed in a linkage that:
to target any biomolecule through their antigen-binding sites.
1. Has a constant leakage of ligand from the matrix that
becomes a contaminant in the purified preparation.
2. Includes a charged isourea group in the linkage,
resulting in nonspecific binding.
3. Causes extensive crosslinking of the matrix, reducing the
ability of large molecules to penetrate into the interior of
the resin.
We offer a number of activated affinity supports that are designed

to couple ligands of every type via stable, uncharged covalent
linkages that avoid introducing undesirable properties into the
supports. The activation chemistry and protocols have been
optimized to ensure excellent coupling yields with minimal effort
under a variety of conditions. Each activated support comes with
instructions for use and literature references as examples. The
associated kits contain all the coupling buffers, wash buffers and
columns necessary to perform the ligand immobilization and
produce a support ready to perform an affinity separation.

Coupling Affinity Ligands through Amine Groups Thermo Scientific UltraLink Biosupport binding capacity for various proteins.
Capacity Protein Coupling Buffer

The most common functional target for immobilizing protein
molecules is the amine group, which is present on the vast 35.0 mg/ml Myoglobin 0.1 M CHES, 1.0 M sodium citrate, pH 9.0
majority of proteins because of the abundance of lysine side chain 21.5 mg/ml Penicillin Acylase 0.1 M sodium phosphate, 1.1 M sodium
ε-amines and N-terminal α-amines. Thermo Scientific AminoLink® sulfate, pH 7.4
Coupling Resin and AminoLink Plus Coupling Resin are prepared 20.9 mg/ml α-chymotrypsin 0.1 M borate, 1.5 sodium sulfate, pH 9.0
from crosslinked agarose supports, and they are designed to 35.5 mg/ml BSA 0.1 M borate, 1.5 sodium sulfate, pH 9.0
create a stable linkage between amine groups and the support
29.8 mg/ml Lysozyme 0.1 M borate, 1.0 sodium sulfate, pH 9.0
material. AminoLink Resins are activated to contain numerous
aldehyde groups, which can be used to immobilize amine-containing 21.0 mg/ml Human IgG 0.1 M borate, 1.5 sodium sulfate, pH 9.0
ligands by reductive amination.
A third option for immobilizing amine-containing affinity ligands
The immobilization reaction using reductive amination involves is the use of carbonyl diimidazole (CDI) to activate hydroxyls on
the formation of an initial Schiff base between the aldehyde and agarose supports to form reactive imidazole carbamates. This
amine groups, which then is reduced to a secondary amine by reactive group is formed on the support in organic solvent and
the addition of sodium cyanoborohydride. The cyanoborohydride stored as a suspension in acetone to prevent hydrolysis. Reaction
reducing agent used during the coupling process is mild enough of the support in an aqueous coupling buffer with primary
not to cleave disulfides in most proteins, and it will not reduce the amine-containing ligands causes loss of the imidazole groups
aldehyde reactants – only the Schiff base intermediates. It is best and formation of carbamate linkages. The coupling process
to avoid stronger reducing agents such as sodium borohydride occurs at basic pH (8.5–10), but it is a slower reaction with
because of the potential for disulfide reduction of the protein and proteins than reductive amination or azlactone coupling. Thermo
reduction of the aldehydes on the support to hydroxyls, effectively Scientific Pierce CDI Supports are available with the CDI-activated
quenching the reaction. Depending on the type and amount of group, and they are particularly adept at immobilizing peptides and
ligand present, a coupling reaction using reductive amination can small organic molecules. The reaction also can be done in
achieve immobilization yields of greater than 85%. organic solvent to permit coupling of water-insoluble ligands.
O H2N–R N
N R H
NaCNBH3 H O N O N
H H2N—R R
O O
Aldehyde Group on
Thermo Scientific Affinity Ligand Coupled
AminoLink Coupling Resin via Secondary Amine Bond
Reactive Imidazole Carbamate
on Thermo Scientific Pierce Affinity Ligand Coupled
CDI Supports via Carbamate Bond
Another amine-reactive strategy that can be used for immobilization
is the azlactone ring present in UltraLink Biosupport. A primary
amine will react with an azlactone group in a ring-opening
process that produces an amide bond at the end of a five-atom
spacer. The group is spontaneously reactive with amines,
requiring no additives or catalysts to drive the coupling process.
The UltraLink Biosupport is supplied dry to ensure stability of the
azlactone groups until use. Adding a quantity of the support to a
sample containing a protein or other amine-containing molecule
causes immobilization to occur within about one hour. For protein
immobilization at high yield, it is recommended that the coupling
buffer contain a lyotropic salt, which functions to drive the protein
molecules toward the bead surface. This brings the hydrophilic
amines close enough to the azlactone rings to react quickly. The
simple nature of coupling affinity ligands to the UltraLink
Biosupport along with its inherently low nonspecific binding makes
it one of the best choices for immobilization.
CH3 O
N H
H2N—R N
CH3 N R
O H
O O
Azlactone Group on
Thermo Scientific Affinity Ligand Coupled
UltraLink Biosupport via Amide Bond
Covalent Coupling of Affinity Ligands
to Chromatography Supports
Coupling Affinity Ligands through Sulfhydryl Groups Thermo Scientific CarboLink™ Coupling Resin contains long spacer
arms that terminate in hydrazide groups. Reaction of the hydrazides
It is often advantageous to immobilize affinity ligands through with aldehydes forms hydrazone linkages, which are a form of
functional groups other than just amines. In particular, the thiol Schiff base displaying better stability than those formed between
group can be used to direct coupling reactions away from active an amine and an aldehyde. The CarboLink Resin can be used to
centers or binding sites on certain protein molecules. Because immobilize glycoproteins, such as antibodies, after periodate
amines occur at many positions on a protein’s surface, it is usually oxidation of the carbohydrate. Coupling antibodies in this manner
difficult to predict where an amine-targeted coupling reaction will specifically targets the heavy chains in the Fc portion of the
occur. However, if sulfhydryl groups that typically are present in molecule. Since this is away from the antigen-binding sites at the
fewer numbers are targeted for immobilization, then coupling can end of the Fv regions, immobilization using this route often results
be done at discrete sites in a protein or peptide. Thiol groups in the best retention of antigen-binding activity.
(sulfhydryls) may be indigenous within a protein molecule or they
can be added through the reduction of disulfides or through the O
Reactive Hydrazide Group
use of various thiolation reagents. Sulfhydryls also can be added on Thermo Scientific NH2
CarboLink Supports
N
to peptide affinity ligands at the time of peptide synthesis by H
adding a cysteine residue at one end of the molecule. This ensures
that every peptide molecule will be oriented on the support in the O
same way after immobilization.
R H
Thermo Scientific SulfoLink® Coupling Resin is designed to
efficiently react with thiol-containing molecules and immobilize
O
them through a thioether linkage. The support contains an iodo- Affinity Ligand
acetyl group at the end of a long spacer arm, which reacts with Coupled via N R
Hydrazone Bond N
sulfhydryls through displacement of the iodine. Optimal conditions H
for the reaction are an aqueous environment at slightly basic pH,
wherein amines are not very reactive toward the iodoacetyl
function, but thiols are highly reactive due to their increased The CarboLink Resin also can be used to couple carbohydrates and
nucleophilicity. The thioether bond that is formed provides a sugars through their reducing ends. Aldehyde- or ketone-containing
stable linkage to any sulfhydryl-containing molecule. sugars will react with the immobilized hydrazide groups without
oxidation of other sugar hydroxyls. However, this reaction may be
Reactive Iodoacetyl Group H
on Thermo Scientific N
dramatically slower than coupling with oxidized sugars because
SulfoLink Supports I these native aldehydes or ketones are usually tied up in acetal or
O ketal ring structures. These rings can open in aqueous solution to
reveal the aldehyde or ketone, but the open structure is present
HS R
only a small percentage of the time. Thus, the reducing ends of
sugars have decreased reactivity toward an immobilized hydrazide,
sometimes requiring days of reaction time to obtain acceptable
Affinity Ligand H immobilization yields.
Coupled via N R
Thioether Bond S Although the hydrazone bond created between the immobilized
O hydrazide and an aldehyde is much more stable than amine-aldehyde
Schiff bases, to obtain a leach-resistant linkage it is recommended
that the Schiff base be reduced with sodium cyanoborohydride.
Coupling Affinity Ligands through Carbonyl Groups This is especially true if a ligand is coupled that has only a single
point of attachment to the support. Reduction of the hydrazone
Most biological molecules do not contain carbonyl ketones or creates a stable bond that will perform well in affinity
aldehydes in their native state. However, it might be useful to chromatography applications.
create such groups on proteins to form a site for immobilization that
directs covalent coupling away from active centers or binding sites.
Glycoconjugates, such as glycoproteins or glycolipids, usually Affinity Ligand
O
contain sugar residues that have hydroxyls on adjacent carbon Coupled via N R
atoms, which can be periodate-oxidized to create aldehydes. Hydrazone Bond N
H
Controlled oxidation using 1 mM sodium meta-periodate at 0°C will
selectively oxidize sialic acid groups to form an aldehyde functionality
on each sugar. Using higher concentrations of periodate (10 mM)
NaCNBH3
at room temperature will result in oxidation of other sugar diols to
create additional formyl groups. Aldehydes on the carbohydrate
portion of glycoconjugates can be covalently linked with O
affinity supports through an immobilized hydrazide, hydrazine or H
Stable Ligand N R
amine group by Schiff base formation or reductive amination. Linkage N
H

Coupling Affinity Ligands through Carboxyl Groups Coupling Affinity Ligands through
The carboxyl group is a frequent constituent of many biological Reactive Hydrogens
molecules. Particularly, proteins and peptides typically contain For molecules containing no easily reactive functional groups,
numerous carboxylic acids due to the presence of glutamic acid, immobilization may be difficult or even impossible using current
aspartic acid and the C-terminal α-carboxylate group. Carboxylic technologies. Certain drugs, steroids, dyes and other organic
acids may be used to immobilize biological molecules through the molecules often have structures that contain no available “handles”
use of a carbodiimide-mediated reaction. Although no activated for convenient immobilization. In other cases, functional groups
support contains a reactive group that is spontaneously reactive that may be present on a molecule have low reactivity or are
with carboxylates, chromatography supports containing amines sterically hindered, prohibiting efficient coupling. Often, these
(or hydrazides) may be used to form amide bonds with carboxyl- compounds that are difficult to immobilize will have certain active
ates. Molecules containing carboxylates may be activated to react (or replaceable) hydrogens that can be condensed with formalde-
with an immobilized amine (or hydrazide) through reaction with the hyde and an amine in the Mannich reaction. Certain hydrogens in
water-soluble carbodiimide EDC. ketones, esters, phenols, acetylenes, α-picolines, quinaldines and
EDC reacts with carboxylates to form an intermediate ester that is other compounds can be aminoalkylated using this reaction.
reactive with nucleophiles such as primary amines. The reaction Formally, the Mannich reaction consists of the condensation of
takes place efficiently between about pH 4.5 and pH 7.5, and it is formaldehyde (or another aldehyde) with ammonia and another
complete within two to four hours, depending on the temperature. compound containing an active hydrogen. Instead of using
The intermediate ester is subject to hydrolysis; therefore, it is ammonia, this reaction can be done with primary or secondary
beneficial if the amine-containing ligand to be immobilized is amines or even with amides.
included in the reaction medium upon addition of EDC, so it can
Use of the Mannich reaction for the preparation of affinity supports
react immediately with the ester as it forms.
offers some unique advantages beyond that of its effective use to
Thermo Scientific CarboxyLink Coupling Resin or the UltraLink immobilize compounds that are difficult to couple. For instance,
DADPA Resin may be used to immobilize carboxylate-containing polymerization is often a problem when using the Mannich reaction
ligands by EDC. CarboxyLink Resin contains a nine atom spacer arm for solution-phase chemistries, especially when multiple reactive
and UltraLink DADPA contains a 12-atom spacer arm to minimize hydrogens are present on a molecule. When one of the reactive
steric hindrance. species is immobilized, the reaction is more controlled and unde-
sirable side reactions are inhibited. Compounds with phenolic
H H O
N N residues (often found in drugs) can be coupled without difficulty.
NH2 + R In addition, the Mannich reaction is a superior alternative to the
OH seldom-used diazonium coupling method. Both the diazonium group
and the resultant diazo linkage are unstable. In contrast, immobili-
Immobilized DADPA on Carboxylate-Containing
Thermo Scientific CarboxyLink Resin Affinity Ligand
zation using Mannich condensations result in very stable covalent
bonds suitable for the most critical affinity separations.
EDC We have developed the Thermo Scientific PharmaLink™ Immobilization

Kit, which is based on the principles of the Mannich reaction.
The PharmaLink Resin included in the kit is immobilized diamino-
H H H dipropylamine (DADPA), which is the source of the primary amine
N N N R for the Mannich reaction. The kits also include coupling buffer,
coupling reagent, wash buffer and accessories.
O
Immobilized Ligand via
Amide Bond Formation
OH
HO
HO
Estradiol-17β
O
+ Formaldehyde
H H H
H H H H N N N
N N NH2
H+, 37˚C
HO
Immobilized DADPA on Immobilized Ligand via
Thermo Scientific PharmaLink Resin Aminoalkyl Bond Formation
Thermo Scientific Products for Covalent Coupling
of Affinity Ligands to Chromatography Supports
AminoLink Plus Immobilization Kits Efficient immobilization of antibodies and other proteins. Percent coupling
efficiency of different proteins to 2 ml of Thermo Scientific AminoLink Plus
and Coupling Resin Coupling Resin using the pH 7.2 coupling protocol.
The simplest and surest method for making an affinity purification Protein Applied Protein Coupled
resin with antibodies or other proteins. Protein (mg/ml) (mg/ml) Percent Coupled
Protein G 4.6 4.0 83
Thermo Scientific AminoLink Plus Coupling Resin and
Mouse IgG 4.7 4.5 96
Immobilization Kits use activated beaded agarose and a robust
coupling chemistry to immobilize proteins and other ligands through Rat IgG 4.7 4.4 93
primary amines (–NH2) to the resin. Once an antibody or other Goat IgG 0.9 0.8 84
ligand is immobilized, the prepared affinity resin can be used for a Human IgG 4.8 4.6 97
variety of purification methods involving batch or column chroma-
Human IgM 0.9 0.8 93
tography. The resin and linkage are stable in binding and elution
conditions typically used in affinity chromatography, enabling pre-
pared resin to be used for at least 10 rounds of affinity purification.
Y
Agarose
O H2N
Bead
Y
The AminoLink Plus Coupling Reaction involves spontaneous +
Y
C
Y
H2N Bead
formation of Schiff base bonds between aldehydes (on the support) H NH2
and amines (on the ligand) and their subsequent stabilization by NH2 Y Y
Y
incubation with a mild reductant (sodium cyanoborohydride; see
Thermo Scientific
more detailed reaction scheme on next page). The entire coupling AminoLink Plus Resin Antibody with Covalently
reaction, called reductive amination, occurs in four to six hours (Aldehyde Activated) Primary Amines Immobilized Antibody
in simple non-amine buffers such as PBS. Coupling efficiency with
Thermo Scientific AminoLink Support immobilization chemistry.
antibodies and typical proteins is generally greater than 85%,
resulting in 1 to 20 mg of immobilized protein per milliliter of References
agarose resin. Beall, A., et al. (1999). J. Biol. Chem. 274(16), 11344–11351.
Nakasato, Y.R., et al. (1999). Clin. Chem. 45, 2150–2157.
Allan, B.B., et al. (2000). Science 289, 444–448.
Highlights: Lu, R., et al. (2000). J. Neurochem. 74, 320–326
• AminoLink Plus Coupling Resin – aldehyde-activated crosslinked
4% beaded agarose
• Ideal for antibodies and other proteins – immobilize molecules
via primary amines (–NH2)
• Flexible coupling conditions – efficient (> 85%) coupling over a
wide range of pH (4–10) and buffer conditions (PBS or other 44894 AminoLink Plus Immobilization Kit Kit
Includes: AminoLink Plus Columns 5 x 2 ml
non-amine buffer with or without organic solvent); regular Neutral pH Coupling Buffer (pH 7.2) 500 ml
(PBS, pH 7.2) and enhanced (borate, pH 10) coupling protocols Enhanced Coupling Buffer (pH 10) 500 ml
Quenching Buffer 60 ml
provided Wash Solution 240 ml
• Stable, permanent immobilization – coupling reaction results in Sodium Cyanoborohydride Solution 0.5 ml
Column Accessories
stable, leak-resistant secondary amine bond between resin
and ligand 20394 AminoLink Plus Immobilization Trial Kit Trial Kit
Includes: AminoLink Plus Column Reagents and Buffers 1 x 2 ml
• Better than immobilization to CNBr-activated agarose – bond is
more stable and uncharged, resulting in less nonspecific binding 20475 AminoLink Plus Micro Immobilization Kit Kit
Sufficient reagents for 10 coupling reactions
in affinity purification procedures using 25–100 µg of protein and 20 affinity purifications.
• Versatile and reusable – prepared affinity resin is adaptable to Includes: AminoLink Plus Spin Columns, 10 each
each containing 400 µl of 25% slurry
column and batch affinity techniques and the resin is reusable Phosphate Buffered Saline 1 pack
for typical applications based on protein binding interactions Quenching Buffer 60 ml
Sodium Cyanoborohydride Solution 0.5 ml
• Convenient kits and product sizes – choose one- or five-column Wash Solution 25 ml
kit containing complete sets of buffers, reagents and versatile Elution Buffer 50 ml
spin/drip columns, or select bulk resin. Bulk quantities are Microcentrifuge Collection Tubes 200 each
available for manufacturing applications 20501 AminoLink Plus Coupling Resin 10 ml

AminoLink Immobilization Kits Efficient immobilization at a variety of pH values. The effect of coupling
buffer pH on percent coupling efficiency of 9.58 mg of human IgG to 2 ml of
and Coupling Resin Thermo Scientific AminoLink Resin using the standard coupling protocol.
Links with primary amines (lysine residues and N-terminus) pH Coupling Efficiency of 9.58 mg Human IgG
on proteins, peptides, antigens or antibodies. 4 91.8%
Thermo Scientific AminoLink Coupling Resin is crosslinked 5 92.7%

4% beaded agarose that has been activated with aldehyde 6 89.1%
groups. Proteins and other molecules with primary amines can 7 87.3%
be covalently attached (immobilized) to AminoLink Resin to make
8* 85.3%
chromatography columns for use in affinity purification. The
aldehyde groups form stable secondary amine bonds with primary 9* 94.9%
amines such as exist in the side chain of lysine (K) residues, which 10* 98.4%
are generally abundant and readily accessible in proteins. Once a
* Schiff-base formation occurs readily at high pH, but reduction to stable secondary
protein is immobilized, the prepared affinity resin can be used for a amine bond requires subsequent incubation with sodium cyanoborohydride (AminoLink
variety of batch and column affinity purification methods involving Reductant) at pH 7.2.
binding interactions with the immobilized protein. The resin and
References
linkage are stable in most binding and elution conditions typically Cheadle, C., et al. (1994). J. Biol. Chem. 269(39), 24034–24039.
used in affinity chromatography, enabling prepared resin to be Cofano, F., et al. (1990). J. Biol. Chem. 265(7), 4064–4071.
used for multiple rounds of affinity purification procedures. DeSilva, B.S. and Wilson, G.S. (1995). J. Immunol. Method 188, 9–19.
Rivero-Lezcano, O.M., et al. (1994). J. Biol. Chem. 269(26), 17363–17366.
Czermak, B.J., et al. (1999). J. Immunol. 162, 2321–2325.
Highlights: Assad, F.F., et al. (2001). J. Cell Biol. 152, 531–543.
• AminoLink Coupling Resin – aldehyde-activated crosslinked Zuk, P.A. and Elferink, L.A. (2000). J. Biol. Chem. 275(35), 26754–26764.6.
4% beaded agarose
• Ideal for antibodies and other proteins – immobilize molecules Ordering Information
via primary amines (–NH2)
• Flexible coupling conditions – efficient (> 85%) coupling over a Product # Description Pkg. Size
wide range of pH (4–10) and buffer conditions (PBS or other 44890 AminoLink Immobilization Kit Kit
non-amine buffer with or without organic solvent) Includes: AminoLink Columns 5 x 2 ml
AminoLink Coupling Buffer (pH 7.0) 240 ml
• Stable, permanent immobilization – coupling reaction results in Quenching Buffer 60 ml
stable, leak-resistant secondary amine bond between resin Wash Buffer 240 ml
and ligand Sodium Cyanoborohydride Solution 0.5 ml
Column Accessories
• Better than immobilization to CNBr-activated agarose – bond is
more stable and uncharged, resulting in less nonspecific binding 20384 AminoLink Immobilization Trial Kit Trial Kit
Includes: AminoLink Column Reagents and Buffers 1 x 2 ml
in affinity purification procedures
20381 AminoLink Coupling Resin 10 ml
• Versatile and reusable – prepared affinity resin is adaptable to
column and batch affinity techniques and the resin is reusable 20382 AminoLink Coupling Resin 50 ml
for typical applications based on protein binding interactions 44892 AminoLink Reductant 2x1g
(Sodium cyanoborohydride)
H2N
H
Y
O N
Agarose
Agarose
Agarose
N
Y
NaCNBH3
Bead
Bead
Bead
Y
Y
C C C Bead
H H H H Y Y
Y
Protein primary amines on antibody react Sodium cyanoborohydride Many aldehyde groups per bead
spontaneously with aldehyde groups on reduces Schiff base to stable and several amines per antibody
resin resulting in Schiff-base bonds secondary amine bond and many antibody molecules per bead
Thermo Scientific AminoLink Support detailed immobilization chemistry.
UltraLink Biosupport Pierce CDI Supports

A durable, polyacrylamide resin, activated for efficient coupling Carbonyldiimidazole-activated resins for ligand immobilization.
of proteins.
Highlights:
Thermo Scientific UltraLink Biosupport is a durable, porous resin that • Reliable coupling chemistry – immobilization occurs through the
is activated to enable efficient and direct covalent immobilization of reaction of N-nucleophiles with 1,1’-carbonyldiimidazole groups
proteins and other biomolecules through their primary amines for use of the resin to form a stable, uncharged N-alkylcarbamate linkages
in affinity purification procedures. • Easy-to-use – no secondary reagents needed; just wash equilibrate
resin in alkaline coupling buffer and add ligand; reaction proceeds
Highlights:
spontaneously
• High coupling efficiency and capacity – immobilizes proteins
• Stable activation – half-life of hydrolysis is much longer than
with very high efficiency and coupling capacity in 1 hour
hydroxysuccinimide ester activations, making immobilization
• Specific and leak-proof coupling chemistry – reacts specifically reactions simpler to prepare and control; simplifies filtration and
with primary amines (–NH2), resulting in amide bonds that are washing before adding a ligand or protein
stable for use in many affinity purification procedures; coupling
• Well-defined coupling conditions – reaction is most efficient with
reaction has no leaving group to contaminate samples
primary amines at pH 9-11
• Easy to use – no pre-swelling or secondary reagents required;
simply weigh the needed amount of dry support and add the
ligand solution to initiate coupling reaction CDI-Activated Crosslinked 6% Beaded Agarose:
• Flexible coupling conditions – perform immobilization reaction • Beaded agarose – the most popular resin for routine affinity
in any of a variety of non-amine buffers and pH levels; coupling purification methods
is most efficient in buffers containing a lyotropic salt such as • Highly activated – at least 50 µmol 1,1’-carbonyldiimidazole (CDI)
sodium citrate; coupling compatible with or without organic solvent groups per milliliter of resin
• Excellent reusability – prepared affinity resin can be used with • Convenient form – supplied as stabilized, 50% slurry in acetone
typical binding and elution procedures for more than 100 cycles
of affinity purification without significant loss of binding capacity CDI-Activated Trisacryl® GF-2000:
• Durable, high-performance resin – porous beads have a 60 µm • Trisacryl resin – rigid, polyacrylamide matrix allows for high flow rates
diameter, can withstand 100 psi (6.9 bar) and allow for linear
• Hydrophilic matrix without charge effects – provides for low
flow-through rates of 3,000 cm/hour
nonspecific binding
CH3 O • Highly activated – at least 50 µmol 1,1’ -carbonyldiimidazole (CDI)
N
Resin
Resin
Bead
Bead
CH2 + H2N Protein N groups per milliliter of resin

H NH
O • Convenient form – supplied as stabilized, 50% slurry in acetone
O O Protein
N
Therm Scientific H
Resin
Resin
Bead
UtraLink Biosupport Amine Amide Bond Formation O

C N + H2N Protein Bead O
C
N Protein
(Azlactone Ring on Bead) Ligand with Ring Opening
O O
Imidazole Amine Carbamate

Thermo Scientific UltraLink Biosupport immobilization chemistry. Carbamate Ligand Linkage
References
1. Ju, T., et al. (2002). J. Biol. Chem. 277, 169–177. CDI immobilization chemistry.
2. Ju, T., et al. (2002). J. Biol. Chem. 277, 178–186.
3. Kornfeld, R., et al. (1998). J. Biol. Chem. 273, 23202–23210. References
4. Liu, L.A. and Engvall, E. (1999). J. Biol. Chem. 274, 38171–38176. 1. Shenoy, S.K., et al. (2001). Science 294, 1307–1313.
2. Richardson, R.T., et al. (2000). J. Biol. Chem. 275, 30378–30386.
Ordering Information 3. Tanaka, M, et al. (2005). PLoS Biology 3, 764–776.
Product # Description Pkg. Size Ordering Information

53110 UltraLink Biosupport (8–10 ml) 1.25 g
53111 UltraLink Biosupport (50 ml) Product # Description Pkg. Size
6.25 g
20259 Pierce CDI (6X) Support 10 ml
28388 BupH Citrate-Carbonate Buffer Packs 10 packs 1,1’-Carbonyldiimidazole activated crosslinked 6%
28386 BupH Citrate-MOPS Buffer Packs 10 packs beaded agarose
Supplied: stabilized in acetone slurry Agarose
hydrated particle size: 45–165 µm
Activation level: > 50 µmol/ml of resin
20377 Pierce CDI Trisacryl Support 50 ml

100
SulfoLink Immobilization Kits and Coupling Resin
+TCEP
Covalent immobilization of sulfhydryl-containing peptides or +TCEP
proteins for affinity purification. 75
Immobilized
Thermo Scientific SulfoLink Coupling Resin is porous, crosslinked
Percent
6% beaded agarose that has been activated with iodoacetyl 50
groups. When incubated with a solution of peptide or protein that
contains reduced cysteine residues, the iodoacetyl groups react -TCEP
specifically and efficiently with the exposed sulfhydryls (–SH) to 25 -TCEP
form covalent and irreversible thioether bonds that permanently
attach the peptide or protein to the resin. The result is a custom-
made affinity resin for purification of antibodies, antigens and other
0
molecules of interest. Peptide A Calcitonin Peptide
Highlights: Improved retention of peptides on Thermo Scientific SulfoLink Resin with TCEP.
• Specific conjugation through sulfhydryl (–SH) groups – the TCEP effectively reduces peptides to maximize immobilization efficiency.
iodoacetyl groups react specifically with sulhydryls to form Two peptides (Peptide A and human calcitonin peptide) were incubated with
25 mM TCEP for 30 minutes and immobilized onto SulfoLink Resin via their reduced
irreversible thioether bonds
sulfhydryl groups. Peptide A’s cysteine had oxidized during long-term storage
• Separate kits optimized for peptides or proteins – kits include and the calcitonin peptide contained an internal disulfide bond. Each peptide also
optimized reagents for preparing peptide or protein samples for contained an amine-terminal fluorescent probe by which the binding of the peptide
efficient immobilization could be monitored during the immobilization and wash steps.
• Fast – spin columns increase protocol speed; prepare and References
couple samples in 2 hours (peptides) to 3.5 hours (proteins) Grunwald, R. and Meissner, G. (1995). J. Biol. Chem. 270(19), 11338–11347.
Seubert, P., et al. (1993). Nature 361, 260–263.
• Flexible coupling conditions – use pH 7.5–9.0 aqueous buffers, Sukegawa, J., et al. (1995). J. Biol. Chem. 270(26), 15702–15706.
organic solvent (e.g., 20% DMSO) or denaturant (guanidine•HCl), Wisniewski, J.R., et al. (1994). J. Biol. Chem. 269(46), 29261–29264.
Sakaguchi, K., et al. (2000). J. Biol. Chem. 275, 9278–9283.
as needed for protein or peptide solubility during coupling reaction Tan, M., et al. (2000). Proc. Natl. Acad. Sci. USA 97, 109–114.
• Easy-to-follow instructions – streamlined protocols for sample Quill, T.A., et al. (2001). Proc. Natl. Acad. Sci. USA 98, 12527–12531.
preparation, immobilization, and affinity purification Tokumaru, H., et al. (2001). Cell 104, 421–432.
Assad, F.F., et al. (2001). J. Cell Biol. 152, 531–543.
• High capacity – immobilize 1–2 mg peptide or 2–20 mg protein
per 2–ml column of SulfoLink Coupling Resin
H
Agarose

Bead
N
I + HS Peptide
44995 SulfoLink Immobilization Kit for Proteins Kit
O Includes: SulfoLink Columns 5 x 2 ml
12-atom Iodoacetyl SulfoLink Preparation Buffer 7.5 ml
Spacer Group SulfoLink Coupling Buffer 500 ml
Wash Solution 120 ml
Phosphate Buffered Saline 1 pack
2-Mercaptoethylamine 5 x 6 mg
Thermo Scientific Reduced Sulfhydryl
L-Cysteine 100 mg
SulfoLink Coupling Resin Molecule Spin Desalting Columns 5 x 5 ml
44999 SulfoLink Immobilization Kit for Peptides Kit

Includes: SulfoLink Columns 5 x 2 ml
SulfoLink Coupling Buffer 120 ml
Wash Solution 120 ml
Phosphate Buffered Saline 1 pack
Bond-Breaker® TCEP 0.5 ml
L-Cysteine 100 ml
H
Agarose
Bead
N 20325 SulfoLink Trial Kit Trial Kit

S Peptide + HI Includes: Pre-packed Column of SulfoLink Resin 1 x 2 ml
O Buffers and Reagents for one protein
or peptide immobilization
Thioether
Bond 20401 SulfoLink Coupling Resin 10 ml
20402 SulfoLink Coupling Resin 50 ml
Immobilized Peptide
Thermo Scientific SulfoLink Support immobilization chemistry.
UltraLink Iodoacetyl Resin UltraLink Iodoacetyl Micro Peptide Coupling Kit

Polyacrylamide resin for coupling sulfhydryl-containing ligands. Easily prepare a small-scale affinity column with sulfhydryl-
containing peptides.
Thermo Scientific UltraLink Iodoacetyl Resin is a durable,
porous resin that is activated to enable efficient and direct The Micro Peptide Coupling Kit is a microcentrifuge spin
covalent immoblization of peptides and other ligands through their column kit for immobilizing small amounts (25–250 µg) of sulfhy-
sulfhydryl groups (–SH) for use in affinity purification procedures. dryl-containing peptides (e.g., cysteine-terminated peptides) onto
The beaded resin is a hydrophilic copolymer of polyacrylamide a beaded porous resin to create a small, reusable affinity column.
and azlactone having a rigid polymeric structure with high surface The coupling and affinity purification procedures are optimized for
area and pore volume. Each azlactone group has been modified to small sample volumes (200–300 µl). Wash and elution steps are
form a 15-atom spacer arm that terminates in an iodoacetyl group, achieved rapidly and efficiently with the convenient microcentrifuge
which is capable of reacting with sulfhydryl groups (e.g., side spin columns. Each kit contains sufficient reagents for 10 coupling
chain of reduced cysteine residues) to covalently immobilize reactions and 20 affinity purifications. The kit is ideal for immobilizing
peptide or other sulfhydryl-containing ligands. The bead structure peptide antigens that containing a terminal cysteine residue for
and efficiently coupling chemistry of Iodoacetyl-Activated use in purifying specific antibodies from small serum, ascites or
UltraLink Support results in high protein binding capacity, high culture supernatant samples.
linear flow rates, low nonspecific binding and overall superior
performance in affinity chromatography. UltraLink Resins are ideal Highlights:
for medium pressure applications such as FPLC. • Optimized for small scale – ideal for coupling small amounts
(25–250 µg) of sulfhydryl-containing peptide or protein for
Highlights: purification of specific antibodies from crude serum
• High coupling efficiency and capacity – immobilizes sulfhydryl- • High-performance affinity resin – uses durable, polyacrylamide-
containing proteins or other ligands with high efficiency and based UltraLink Iodoacetyl Resin for specific reaction to
coupling capacity in 1 hour sulfhydryl groups
• Specific and leak-proof coupling chemistry – reacts specifically • Efficient coupling chemistry – immobilization efficiency > 85%
with sulfhydryl groups (reduced thiols), resulting in thioether (as measured with 1 hour reaction using insulin, calcitonin and
bonds that are stable for use in many affinity purification procedures osteocalcin peptides)
• Simple coupling conditions – perform immobilization reaction in • Fast and easy to use – perform wash and elution steps using
any of a variety of buffers; coupling is most efficient and specific a microcentrifuge
at pH 8.0–8.5; coupling compatible with or without organic solvent • Reusable – use prepared peptide resin several times with no
• Excellent reusability – prepared affinity resin can be used with significant loss of capacity
typical binding and elution procedures for many cycles of affnity
purification without significant loss of binding capacity
• Durable, high-performance resin – porous beads have a 60 µm Ordering Information
diameter, can withstand 100 psi (6.9 bar) and allow for linear
flow-through rates of 3,000 cm/hour
20485 UltraLink Iodoacetyl Micro Peptide Coupling Kit Kit
References Sufficient materials to couple 10 sulfhydryl containing
1. Ju, T., et al. (2002). J. Biol. Chem. 277, 169–177. peptides or protein and perform 20 affinity purifications.
2. Hill, K., et al. (2000). J. Biol. Chem. 275(6), 3741–3744. Includes: UltraLink Iodoacetyl Resin Spin Columns 10 each
3. Liu, L.A. and Engvall, E. (1999). J. Biol. Chem. 274, 38171–38176. Each column contains 400 ml of 25% slurry
4. Bicknell, A.B., et al. (2001). Cell 105, 903–912. Coupling Buffer 100 ml
L-Cysteine•HCI 100 mg
Wash Solution 25 ml
Ordering Information Phosphate Buffered Saline 1 pack
IgG Elution Buffer 50 ml
Microcentrifuge Collection Tubes 200 each
53155 UltraLink Iodoacetyl Resin 10 ml
Support: UltraLink Biosupport
Thermo Scientific
Thermo Scientific
H
UltraLink Bead
H
UltraLink Bead
N N
I + HS Peptide S Peptide + HI
O O
15-atom Iodoacetyl Thioether
Spacer Group Bond
Iodoacetyl-Activated Reduced Sulfhydryl Immobilized Peptide

Thermo Scientific UltraLink Resin Molecule
Thermo Scientific UtraLink Iodoacetyl immobilization chemistry.

Disulfide Reducing Agents Ellman’s Reagent and Sulfhydryl Addition Kit
Reduce disulfide bonds to produce sulfhydryl groups for Measure and add free sulfhydryls to ensure success of
immobilization on Thermo Scientific SulfoLink or UltraLink Resins. cysteine-targeted immobilization.
Free sulfhydryls are required for immobilization onto sulfhydryl- Ellman’s Reagent, also called DTNB, is a versatile water-soluble
reactive affinity supports. Cysteines in proteins and peptides compound for quantifying free sulfhydryl groups in solution. A
usually exist as cystines (disulfide bridges) and must be reduced solution of this compound produces a measurable yellow-colored
to expose sulfhydryls for coupling. Reduction can be accomplished product when it reacts with sulfhydryls (–SH groups). By testing
with free or immobilized reducing agents. Free reducing agents an unknown sample, such as a peptide having a terminal cysteine
are efficient in reducing all disulfides in proteins, including those residue, compared to a standard curve made with known amounts
buried in the tertiary structure, but they must be removed from the of free, reduced cysteine (Product # 44889), availability of reduced
reduced sample with a desalting column before coupling to the sulfhydryls in the sample can be determined.
support. Immobilized reducing agents enable reduction of disulfides
and simple removal of the reduced sample from the reducing The Sulfhydryl Addition Kit provides the essential reagents and
agent. This is especially helpful when reducing peptides whose procedure for creating new sulfhydryl groups on a protein or
small size prevents them from being effectively desalted. other molecule that contains available primary amines (–NH2).
The kit uses SATA reagent, which forms covalent bonds to primary
+NH Cl– amines. The result is addition of a stable (capped) sulfhydryl
OH 3
HS group, which can later be exposed by gentle treatment with
O
hydroxylamine, making the molecule ready for conjugation to
O 2-Mercaptoethylamine•HCI SulfoLink Coupling Resin, UltraLink Iodoacetyl Resin or other sulf-
MW 113.61
+ – hydryl-reactive immobilization method.
HO P H Cl
OH
HS
SH
O OH
OH 23460 Sulfhydryl Addition Kit Kit
Adds free sulfhydryl groups to proteins.
TCEP•HCl DTT Includes: SATA 2 mg
MW 286.65 MW 154.25 Hydroxylamine•HCl 5 mg
10X Conjugation Buffer Stock 20 ml
Phosphate Buffered Saline Pack 1 pack
Ordering Information Dimethylformamide (DMF) 1 ml
Dextran Desalting Column 1 x 5 ml
Column Extender 1
Product # Description Pkg. Size Ellman’s Reagent (DTNB) 2 mg
20408 2-Mercaptoethylamine•HCl 6 x 6 mg Cysteine•HCl H2O 20 mg
20290 DTT, Cleland’s Reagent 5g 22582 Ellman’s Reagent 5g

(Dithiothreitol) (5,5’-Dithio-bis-[2-nitrobenzoic acid])
20291 Dithiothreitol (DTT) in No-Weigh™ Format 48 tubes 44889 Cysteine•HCl 5g

7.7 mg DTT/tube. Makes 100 µl of 0.5 M DTT.
20490 TCEP•HCl 1g
(Tris[2-carboxyethyl]phosphine hydrochloride)
20491 TCEP•HCl 10 g
77720 Bond-Breaker TCEP Solution, Neutral pH 5 ml
77712 Immobilized TCEP Disulfide Reducing Gel 5 ml
CarboLink Immobilization Kit and Coupling Resins

O
Immobilize polyclonal antibodies and other glycoproteins through
carbohydrate groups. O H
Agarose
Bead
NH2
N
H
Antibody with Carbohydrate
Thermo Scientific CarboLink 23-atom Hydrazide Groups Oxidized to Form
Coupling Resin and Kits Spacer Group Aldehyde Groups
provide for covalent immobi-
lization of glycoproteins and Thermo Scientific
other carbohydrate-containing CarboLink Resin Oxidized Glycoprotein
molecules to beaded agarose
(or polyacrylamide UltraLink
Support) for use in affinity
purification procedures.
Carbohydrate moieties in O
Agarose
N
Bead
glycoproteins contain common N
sugars whose cis-diol groups are easily oxidized with sodium H
meta-periodate (included in the CarboLink Kit) to yield aldehydes. Hydrazone
Bond
When incubated with the CarboLink Resin, these aldehyde groups
react spontaneously with the hydrazide group of the activated
Immobilized Glycoprotein
resin to form stable, covalent bonds. The immobilization strategy is
especially useful for glycoproteins, such as polyclonal antibodies,
because it allows attachment of the molecule at domains that will Thermo Scientific CarboLink Support immobilization chemistry.
not interfere with binding sites that are critical for the intended
affinity purification. Once a molecule is coupled, the prepared References
affinity resin can be used multiple times in typical protein affinity 1. Kumar, P.G., et al. (2001). J. Biol. Chem. 276, 41357–41364.
2. Strakova, Z., et al. (1997). Mol. Pharmacol. 51, 217–224.
purification procedures. 3. Brown, M.A., et al. (2000). J. Biol. Chem. 275, 19795–19802.
4. Butko, P., et al. (1999). J. Immunol. 163, 2761–2768.
Highlights: 5. Sequra, M., et al. (1999). Infect. Immun. 67(9), 4646
• CarboLink Coupling Resin – hydrazide-activated crosslinked
6% beaded agarose (or hydrazide-activated UltraLink Support, Ordering Information
a beaded, polyacrylamide resin)
• Efficient immobilization – couple 1–5 mg of oxidized polyclonal Product # Description Pkg. Size
antibody or other glycoprotein per milliliter of resin (CarboLink 44910 CarboLink Immobilization Kit Kit
Resin contains greater than 14 µmol hydrazide groups per milliliter) Includes: CarboLink Columns 5 x 2 ml
CarboLink Coupling Buffer 250 ml
• Stable linkage – resonance structure of the hydrazone bonds CarboLink Wash Buffer 100 ml
are sufficiently stable to allow multiple rounds of affinity CarboLink Oxidant 5 x 5 mg
Spin Desalting Columns 5 x 5 ml
purification with one batch of prepared resin; no stabilizing
reductant required 20355 CarboLink Trial Kit Trial Kit
Sufficient reagents and buffers for
• Flexible and gentle coupling conditions – immobilization reaction preparing 1 x 2 ml immunoaffinity column.
completed in simple buffers (PBS or other non-amine buffer with
20391 CarboLink Coupling Resin 10 ml
or without organic solvent) at near-neutral pH
• Ideal for polyclonal antibodies – immobilizes IgG through 53149 UltraLink Hydrazide 10 ml
carbohydrates in the Fc region, so both antigen binding sites are Spacer Arm: 22 atom
free to interact with the antigen in the mobile phase Capacity: ≥ 15 µmol functionality/ml of resin
• Effective for any molecule with oxidizable sugars – first step is 20504 Sodium meta-Periodate 25 g
oxidation of the sugar groups, which allows the cis-diols of the
IgG to be transformed into reactive aldehyde moieties; these
aldehydes then combine with hydrazide groups on the matrix to
form stable, leak-resistant linkages
• Convenient kits and product sizes – choose one- or five-column
kit containing complete sets of buffers, oxidizing reagent and
versatile spin/drip columns containing the beaded agarose resin;
or choose the polyacrylamide-based UltraLink Support. Bulk
quantities are available for manufacturing applications

CarboxyLink Immobilization Kits and H H
HO Peptide
Agarose
N NH2
Bead
N
Coupling Resins +
O
Immobilize peptides via carboxyl groups to create an affinity column. Immobilized DADPA Carboxyl Ligand
(Thermo Scientific CarboxyLink Resin) (e.g., peptide C-terminus)
Thermo Scientific CarboxyLink Coupling Resin and Kits provide for
covalent immobilization of peptides or other carboxyl-containing
(–COOH) molecules to a porous, beaded resin for use in affinity
purification procedures. CarboxyLink Resin is crosslinked beaded EDC Crosslinker
agarose (or polyacrylamide UltraLink Support) that has been
activated with diaminodipropylamine (DADPA) to contain long
H H H
Agarose
spacer arms, each with a primary amine at the end. When incubated N N N Peptide
Bead
with the resin and the carbodiimide crosslinker EDC (included in
the CarboxyLink Immobilization Kit), carboxyl-containing molecules O
become permanently attached to the support by stable amide
Covalently Immobilized Ligand
bonds. Once a molecule is coupled, the prepared affinity column (attached by amide bond and long spacer arm)
can be used multiple times in typical protein affinity purification
procedures. CarboxyLink Coupling Resins can also be used to Thermo Scientific CarboxyLink Support immobilization chemistry.
immobilize other kinds of molecules using alternative amine-reactive
crosslinking chemistries. Reference
Yoo, B.C., et al. (2002). J. Biol. Chem. 277, 15325–15332.
Highlights:
• CarboxyLink Coupling Resin – DADPA-activated crosslinked Ordering Information
4% beaded agarose (or DADPA-activated UltraLink Support,
a beaded polyacrylamide resin) Product # Description Pkg. Size
• Efficient immobilization – couple 1–2 mg of peptide per milliliter 44899 CarboxyLink Immobilization Kit Kit
of resin (CarboxyLink Agarose Resin activated with greater than Includes: DADPA Columns 5 x 2 ml
16 µmol amine milliliter of resin; DADPA on UltraLink Support EDC 5 x 60 mg
Coupling Buffer 500 ml
activated with greater than 40 µmol amine milliliter of resin) Wash Buffer 120 ml
• Stable linkage – immoblization results in covalent attachment of Accessories
carboxyl groups by amide bonds, allowing for multiple rounds of 20266 CarboxyLink Coupling Resin 25 ml
affinity purification with one batch of prepared resin Support: Crosslinked 4% beaded agarose
Loading: 16–20 µmol available amino groups/ml of resin
• Flexible and gentle coupling conditions – immobilization reaction
completed in simple MES or other non-amine and non-carboxyl, 53154 Carboxylink Immobilization Kit Kit
with UltraLink Resin
near-neutral buffer, with or without organic solvent. Includes: UltraLink DADPA Columns 5 x 2 ml
• Ideal for unmodified peptides – immobilizes peptides with high EDC 5 x 60 mg
Coupling Buffer 500 ml
capacity and various orientations without steric hindrance, Wash Buffer 120 ml
allowing for effective use in affinity purification of specific Accessories
antibodies 22980 EDC 5g
• Convenient kits and product sizes – choose five-column kits with 1-Ethyl-3-[3-dimethylaminopropyl]carbodiimide
hydrochloride
complete sets of buffers, crosslinker and versatile spin/drip
columns containing either type of resin (agarose or 28390 BupH MES Buffered Saline Packs 10 pack
polyacrylamide) or choose stand-alone resin for other uses
PharmaLink Immobilization Kit OH

H O
Immobilize drug and metabolite ligands that contain no easily H H
Molecule with
reactive functional groups. Active Hydrogens Formaldehyde
H
The Thermo Scientific PharmaLink Immobilization Kit uses the H H
Agarose
Bead
Mannich reaction to conjugate active hydrogen chemical groups N N NH2
Mannich Reaction
of ligands to beaded agarose resin for use in affinity purification
procedures. Many small metabolites and drug molecules do not DADPA Resin
contain available primary amines, carboxyl, sulfhydryl or other H20
functional groups that can be easily targeted for chemical conju-
gation to a chromatography support. However, if the compound
contains phenolic or other moieties having active hydrogens, it
can be conjugated to PharmaLink Coupling Resin by condensation HO
with primary amines of the support using the formaldehyde-con- H H H
Agarose
Bead
N N N
taining PharmaLink Coupling Reagent. Once a molecule is coupled, H
the prepared affinity column can be used multiple times in typical
H
affinity purification procedures, such as to purify ligand-specific
OH
antibodies from sera of immunized animals. H H H
Agarose
Bead
N N N
PharmaLink Coupling Resin is crosslinked beaded agarose that has
been activated with diaminodipropylamine (DADPA) to contain long Possible Immobilization Products
spacer arms, each with a primary amine at the end. The Mannich
reaction consists of the condensation of formaldehyde (or other Thermo Scientific PharmaLink Support immobilization chemistry.
aldehyde) with ammonia and a compound containing an active
hydrogen atom. Primary or secondary amines can be substituted
O O O O
for ammonia in the reaction. In the PharmaLink Kit, the primary C C H C C H C C H
HC C H
amine (–NH2) at one end of the immobilized DADPA molecule R O HO R
substitutes for the ammonia reactant, and an active hydrogen in
the target molecule provides the other reactant for the Mannich
reaction. The PharmaLink Coupling Reagent supplies the required O H
C
formaldehyde condensation reagent. N C H N C C H R C C H
N
Highlights:
• PharmaLink Coupling Resin – DADPA-activated crosslinked
4% beaded agarose OH
H
• High-efficiency coupling – PharmaLink (DADPA) Resin is N C H R O H R S H
activated with greater than 16 µmol amine per milliliter of resin
• Stable linkage – immobilization by the Mannich reaction results H
in covalent attachment of ligand, allowing for multiple rounds of
affinity purification with prepared column Thermo Scientific PharmaLink Support immobilization targets. Examples of
• Flexible coupling conditions – coupling buffer is MES-buffered active hydrogen functional groups that can participate in the Mannich reaction
(PharmaLink Immobilization).
saline, pH 4.7, and ethanol can be used to maintain ligand
solubility during the immobilization reaction
Reference
• Immobilizes molecules with active hydrogen groups – couple
1. Rao, M.N., et al. (1997). J. Biol. Chem. 272, 24455–24460.
ligand containing ketones, esters, phenols, acetylenes,
α-picolines, quinaldines and other groups
• Ideal for immobilizing small metabolites and drug compounds – Ordering Information
use for steroidal compounds, dyes and other organic molecules
that contain no available “handles” for easy immobilization, Product # Description Pkg. Size
or that have functional groups with low reactivity or that are
44930 PharmaLink Immobilization Kit* Kit
sterically hindered Includes: PharmaLink Columns 5 x 2 ml
PharmaLink Coupling Buffer 50 ml
PharmaLink Coupling Reagent 4 ml
PharmaLink Washer Buffer 240 ml
Accessories
77168 PharmaLink Coupling Reagent 4 ml

* See patent information on inside back cover.

Reference
Pierce Maleic Anhydride Plates Brett, P.J., et al. (2002). J. Biol. Chem. 277, 20468–20476.
Proteins and other primary amine-containing compounds covalently

attach to the microplate. Ordering Information
Great for immobilization of compounds that do not normally stick to Product # Description Pkg. Size
plain polystyrene plates.
15110 Maleic Anhydride Activated 5 plates
Clear Polystyrene 96-Well Plates
Highlights:
• Spontaneously reacts with primary amines 15112 Maleic Anhydride Activated 25 plates
Clear Polystyrene 96-Well Plates
• Maleic anhydride retains its integrity and coupling availability
for months Clear Polystyrene 8-Well Strip Plates
• Available as standard 96-well microplates and as 12 × 8-well
strip plates Clear Polystyrene 8-Well Strip Plates
• Each well activated with 200 µl reagent 15108 Maleic Anhydride Activated 5 plates
• Plates tested for specific signal:noise ratio and coefficient of White Polystyrene 96-Well Plates
variation (CV) to ensure consistent performance
• Approximate binding capacity: 125 pmol biotin-pentylamine
per well
H2N Peptide H2N Peptide
O O H
O N O OH
O O -O Peptide HO
O O pH 8-9 pH 3-4
O O
Maleic Anhydride Immobilized Peptide Hydrolyzed Product

Activated Plate (ready for use in assay at pH > 7) (peptide released)
Reaction scheme for coupling amine-containing molecules to Maleic

Anhydride Plates.
Pierce Maleimide Activated Plates Reaction scheme for coupling sulfhydryl-containing molecules to
maleimide-activated plates.
A convenient alternative to amine-reactive chemistries for
HS Peptide
attaching sulfhydryl-containing compounds.
tide
Pep
S
Maleimide groups specifically and covalently conjugate sulfhydryl O O
N
groups at neutral pH, creating a stable thioether bond. O N O pH 6.5-7.5 O N O
Highlights:
• Spontaneous immobilization of peptides containing reduced
Maleimide Activated Plate Immobilized Peptide
terminal cysteines or proteins with free sulfhydryls
• Available in clear, white and black 96-well plates
• Each well activated with 100 µl maleimide reagent and blocked
with 200 µl bovine serum albumin (BSA)
• Plates tested for specific signal:noise ratio and coefficient of Ordering Information
variation (CV) to ensure consistent performance
• Approximate binding capacity: 100–150 pmol sulfhydryl-peptide Product # Description Pkg. Size
per well 15150 Maleimide Activated 5 plates
Clear Polystyrene 8-Well Strip Plates
15152 Maleimide Activated 5 plates
White Polystyrene 96-Well Plates
15153 Maleimide Activated 5 plates
Black Polystyrene 96-Well Plates

Antibody immobilization: choosing the best Thermo Scientific Support.
Orientation Kits
AmnioLink Plus UltraLink CarboLink SulfoLink (Protein A or Protein G)
Coupling Resin Biosupport Coupling Resins Coupling Resins See page 61
Monoclonal Antibodies Advantages: Advantages: Advantages: Advantages: Advantages:
• Good choice when • Good choice if antibody • Correctly orients antibody • Good choice for • Allows for correct
only small amounts of can withstand 1.0 M • Antibody must be able antibodies that have orientation of antibodies
antibody are available sodium citrate or sulfate to withstand oxidation extremely high avidity • Gentle coupling conditions
• Couple over a broad • High capacity conditions for their antigen • Either Protein A or G will
pH range • Fast, efficient coupling • Good for antibodies with • Allows for gentle elution bind most antibodies
• Good coupling efficiency • Good, for large-scale or low avidity for antigen conditions
fast-flow applications Disadvantages:
Disadvantages: Disadvantages: Disadvantages: • If purifying antigen from
• Reduction of Schiff’s base Disadvantages: • Not all monoclonals have • Must first reduce antibody serum, antibodies may
with sodium cyanoborohy- • Some antibodies may be carbohydrate accessible prior to coupling bind to Protein A or G and
dride may adversely affect coupled through antigen- for coupling • Not good for antibodies co-purify with antigen
monoclonals binding site • Conditions necessary for with low affinity for • Crosslinking results in
• Some antibodies may • Some antibodies may pre- coupling may adversely their antigens some loss of antibody
be coupled through anti- cipitate in high-salt buffer affect some monoclonals activity
gen-binding site
Polyclonal Antibodies Advantages: Advantages: Advantages: Advantages: Advantages:
• Excellent coupling • Good choice for • Correctly orients antibody • Good choice for antibod- • Allows for correct
efficiency large-scale or fast-flow • Antibody must be able ies that have avidity for orientation of antibodies
• Good antigen recovery applications to withstand oxidation their antigen • Gentle couple conditions
• High capacity conditions • Allows for gentle elution • Either Protein A or G will
Disadvantages: • Fast, efficient coupling • Good for antibodies with conditions bind most antibodies
• Some antibodies may low avidity for antigen
be coupled through anti- Disadvantages: Disadvantages: Disadvantages:
gen-binding site • Some antibodies may Disadvantages: • Must first reduce antibody • If purifying antigen from
be coupled through anti- • Conditions necessary for prior to coupling serum, antibodies may
gen-binding site coupling may adversely • Not good for antibodies bind to Protein A or G and
• Some antibodies may affect some antibodies with low affinity for their co-purify with antigen
precipitate in high-salt antigens • Crosslinking results in
buffer some loss of antibody
activity
High-Activity Antibodies Advantages:
• Immobilization of reduced
antibody allows for gen-
tler elution conditions
Low-Activity Antibodies Advantages: Advantages:
• Correctly orients antibody • Allows for correct
orientation of antibodies
Disadvantages: • Gentle couple conditions
• Conditions necessary for • Either Protein A or
coupling may adversely Protein G will bind
affect some monoclonals most antibodies
Disadvantages:
• If purifying antigen from
serum, antibodies may
bind to or Protein G and
co-purify with antigen
• Crosslinking results in
some loss of antibody
activity
Avidin:Biotin Binding
Biotin-Binding Proteins
Avidin – The extraordinary affinity of avidin for biotin allows
biotin-containing molecules in a complex mixture to be discretely
bound with avidin. Avidin is a glycoprotein found in the egg white
and tissues of birds, reptiles and amphibians. It contains four identi-
cal subunits having a combined mass of 67,000–68,000 daltons.
Each subunit consists of 128 amino acids and binds one molecule
of biotin. The extent of glycosylation on avidin is high; carbohy-
drate accounts for about 10% of the total mass of the tetramer.
Avidin has a basic isoelectric point (pI = 10–10.5) and is stable over
a wide range of pH and temperature. Extensive chemical
modification has little effect on the activity of avidin, making it
especially useful for protein purification. However, because of
its carbohydrate content and basic pI, avidin has relatively high
nonspecific binding properties.
Streptavidin – Another biotin-binding protein is streptavidin, which

is isolated from Streptomyces avidinii and has a mass of 75,000
Biotin daltons. In contrast to avidin, streptavidin has no carbohydrate and
has a mildly acidic pI (5.5). Thermo Scientific Pierce Streptavidin
Biotin, also known as vitamin H, is a small molecule (MW 244.3) is a recombinant form having a mass of 53,000 daltons and a
that is present in tiny amounts in all living cells. The valeric near-neutral pI. Streptavidin is much less soluble in water than
acid side chain of the biotin molecule can be derivatized to avidin. There are considerable differences in the composition of
incorporate various reactive groups that are used to attach biotin avidin and streptavidin, but they are remarkably similar in other
to other molecules. Once biotin is attached to a molecule, the respects. Streptavidin is also a tetrameric protein, with each
molecule can be affinity-purified using an immobilized version of subunit binding one molecule of biotin with affinity similar to that of
any biotin-binding protein. Alternatively, a biotinylated molecule avidin. Guanidinium chloride will dissociate avidin and streptavidin
can be immobilized through interaction with a biotin-binding protein, into subunits, but streptavidin is more resistant to dissociation.
then used to affinity-purify other molecules that specifically Streptavidin contains an RYD sequence similar to the RGD
interact with it. We offer biotin-labeled antibodies and a number sequence that binds cell surface receptors. The RYD sequence
of other biotinylated molecules, as well as a broad selection of can cause background in some applications.
biotinylation reagents to label any protein.
Thermo Scientific NeutrAvidin Protein – We also offer a degly-
Valeric Acid Side Chain
cosylated version of avidin, known as NeutrAvidin Protein, with a
O
H H
mass of approximately 60,000 daltons. As a result of carbohydrate
O removal, lectin binding is reduced to undetectable levels, yet bio-
H tin-binding affinity is retained because the carbohydrate is
HO not necessary for this activity. NeutrAvidin Protein offers the
S H advantages of a near-neutral pI (6.3) to minimize nonspecific
Biotin
adsorption, along with lysine residues that remain available for
MW 244.3 derivatization or conjugation. NeutrAvidin Protein yields the
lowest nonspecific binding among the known biotin-binding
proteins due to its near-neutral pI and lack of both carbohydrate
and RYD sequence.

Strength of Avidin-Biotin Interaction – The avidin-biotin complex Biotin-Binding Products
is the strongest known noncovalent interaction (Ka = 1015 M-1)
between a protein and ligand. The bond formation between biotin Each of the four biotin-binding proteins discussed is available
and avidin is rapid and, once formed, is unaffected by extremes in a variety of immobilized formats. The support resin used for
of pH, temperature, organic solvents and most denaturing agents. Immobilized Avidin, Streptavidin and NeutrAvidin Protein is a
These features of avidin – features that are shared by streptavidin crosslinked 6%, beaded agarose. Immobilized Monomeric Avidin
and NeutrAvidin Protein – make immobilized forms of the biotin- uses a crosslinked 4% beaded agarose. UltraLink Biosupport is
binding proteins particularly useful for purifying biotin-labeled a durable, polyacrylamide-based resin with a high surface area,
proteins or other molecules. However, the strength of the large pore volume and low nonspecific binding. It is suitable for
interaction and its resistance to dissociation make it difficult to pressures up to 100 psi and linear flow rates up to 3,000 cm/hour.
elute bound proteins from an immobilized support. Harsh, denatur- A biotin-binding protein immobilized on beaded agarose or
ing conditions (8 M guanidine•HCl, pH 1.5 or boiling in SDS-sample UltraLink Biosupport can be used for affinity purification in a
loading buffer) are required to efficiently dissociate avidin:biotin column or batch method. NeutrAvidin Protein and Streptavidin are
complexes. Such conditions damage the support irreversibly so also available bound to polystyrene microplates along with a dried
that it cannot be reused, and denature the eluted proteins so that blocking buffer. These 96-well plates are offered in transparent,
they do not maintain any biological activity. white or black plates to accommodate a variety of assay types.
The plates come in two forms – regular and high-binding capacity.
Because of these binding and elution properties, purifications The high-binding capacity plates contain more of the immobilized
based on avidin:biotin affinity are reserved primarily for small- NeutrAvidin Protein or Streptavidin and are ideal for binding large
scale procedures involving immediate analysis of the eluted amounts of small, biotin-containing molecule (e.g., a biotinylated
sample by reducing SDS-PAGE or other denaturing method. On the peptide). Streptavidin immobilized on MagnaBind Magnetic Beads
other hand, it is possible to take advantage of the strong avidin: is an excellent tool for cell-sorting applications.
biotin binding properties in immunoprecipitation (IP) and pull-down
procedures because the immunoprecipitated “prey” protein can
be recovered using elution conditions that will not A Comparison of Biotin-Binding Proteins
also elute the biotinylated antibody or “bait” protein. In some
The strong association between avidin and biotin can be used in
situations, it may be most appropriate to use a cleavable
the field of affinity separations. By attaching avidin to a solid
biotinylation reagent to label the target molecule so that it may
support, a biotinylated product can be anchored to the same solid
be recovered from its bound state to immobilized avidin by
support. The attachment is stable over a wide range of pH, salt
specific cleavage of the spacer arm between biotin and target
concentrations and temperatures. To dissociate biotin from avidin,
molecule rather than by elution of biotin from avidin.
8 M guanidine•HCl, pH 1.5 or boiling in SDS-PAGE sample buffer
Monomeric Avidin – Immobilized Monomeric Avidin was must be used.
developed to allow the purification of fully functional biotinylated Thermo Scientific
Avidin Streptavidin NeutrAvidin Protein
proteins. Unlike other biotin-binding proteins that require harsh,
denaturing conditions to elute and recover bound molecules, Molecular Weight 67K 53K 60K
Monomeric Avidin binds reversibly to biotin and allows gentle Biotin-binding Sites 4 4 4
elution and recovery of biotinylated molecules using a solution of Isoelectric Point (pl) 10 6.8–7.5 6.3
2 mM biotin to compete for the biotin-binding sites. This makes it
Specificity Low High Highest
possible to harness the avidin:biotin interaction as a purification
tool to recover functional proteins and other biological molecules. Affinity for Biotin (Kd) 10 15 M 10 15 M 10 15 M
Nonspecific Binding High Low Lowest
Thermo Scientific Products for Avidin:Biotin Binding
Immobilized Avidin Products Immobilized Streptavidin Products

Strong biotin interaction creates a nearly irreversible bond. Same high biotin-binding affinity as avidin with low nonspecific
binding.
Immobilized avidin can be used in a variety of applications for
the affinity purification of biotinylated macromolecules. In one Applications:
variation, an antibody that has an affinity for a particular antigen • Purification of membrane antigens in conjunction with
is labeled with biotin. Cells containing the antigen are lysed, then biotinylated monoclonal antibodies1,2
incubated with the biotinylated antibody to form a typical anti- • Cell-surface labeling with biotinylation reagents, followed by
gen/antibody complex. To isolate the antigen, the crude mixture precipitation with immobilized streptavidin3
is passed through an immobilized avidin or streptavidin column, • Purification of cell-surface glycoproteins using biotinylated
which will bind the complex. After appropriate washes, the antigen Concanavalin A4
can be eluted from the column with a low pH elution buffer. The
• Recovery of single-stranded DNA for dideoxy sequencing5
biotinylated antibody is retained by the column.
References
Applications: 1. Gretch, D.R., et al. (1987). Anal. Biochem. 163, 270–277.
• Binding biotinylated anti-transferrin for purifying transferrin 2. Updyke, T.V. and Nicolson, G.L. (1984). J. Immunol. Method 73, 83–95.
3. Lisanti, M.P., et al. (1989). J. Cell Biol. 109, 2117–2127.
from serum1 4. Buckie, J.W. and Cook, G.M. (1986). Anal. Biochem. 156(2), 463–472.
• Binding biotinylated peptides and elution with an SDS/urea solution2 5. Baqui, M., et al. (2003). J. Biol. Chem. 278, 1206–1211.
6. Ellerbroek, S.M., et al. (2001). J. Biol. Chem. 276, 24833–24842.
• Hybridization of biotinylated RNA to its complementary DNA 7. Huh, K-H. and Wenthold, R.J. (1999). J. Biol. Chem. 274, 151–157.
and binding to immobilized avidin, with subsequent elution of the 8. Kilic, F., et al. (2000). Proc. Natl. Acad. Sci. USA 97, 3106–3111.
9. Lesa, G.M., et al. (2000). J. Biol. Chem. 275, 2831–2836.
single-stranded DNA3 10. Liu, L.A. and Engvall, E. (1999). J. Biol. Chem. 274, 38171–38176.
• Purification of double-stranded DNA4
References Ordering Information
1. Wilchek, M. and Bayer, E.A. (1989). Protein Recognition of Immobilized Ligands.
Hutchins, T.W., ed. Alan R. Liss, Inc., pp. 83–90. Product # Description Pkg. Size
2. Swack, J.A., et al. (1978). Anal. Biochem. 87, 114–126.
3. Manning, J., et al. (1977). Biochemistry 16, 1364–1370. 20347 Streptavidin Agarose Resin 2 ml
4. Pellegrini, M., et al. (1977). Nucleic Acids Res. 4, 2961–2973. Support: Crosslinked 6% beaded agarose
5. Claypool, S.M., et al. (2002). J. Biol. Chem. 277, 28038–28050. Capacity: 1–3 mg biotinylated BSA/ml resin
6. Sharma, K.K., et al. (2000). J. Biol. Chem. 275, 3767–3771. 15–28 µg biotin/ml resin
20349 Streptavidin Agarose Resin 5 ml

Ordering Information Support and Capacity: Same as above
20353 Streptavidin Agarose Resin 10 ml

Product # Description Pkg. Size Support and Capacity: Same as above
20219 Avidin Agarose Resin 5 ml 20351 Streptavidin Agarose Columns 5 x 1 ml

Support: Crosslinked 6% beaded agarose Support and Capacity: Same as above
Capacity: ≥ 20 µg biotin/ml resin
53113 Streptavidin UltraLink Resin 2 ml
20225 Avidin Agarose Resin 5 x 5 ml Support: UltraLink Biosupport
Support and Capacity: Same as above Capacity: ≥ 2 mg biotinylated BSA/ml resin
≥ 24 µg biotin/ml resin
20362 Avidin Agarose Columns 5 x 1 ml
Support and Capacity: Same as above 53114 Streptavidin UltraLink Resin 5 ml
Support and Capacity: Same as above
53116 Streptavidin Plus UltraLink Resin 2 ml

Capacity: ≥ 4 mg biotinylated BSA/ml resin
≥ 48 µg biotin/ml resin
53117 Streptavidin Plus UltraLink Resin 5 ml

20357 High Capacity Streptavidin Agarose Resin 2 ml

Support: Crosslinked 6% beaded agarose
Capacity: > 10 mg biotinylated BSA/ml of resin


21344 MagnaBind Streptavidin Beads 5 ml

Support: 1–4 µm, iron oxide particles
Capacity: 2 µg biotin/ml beads

References
Immobilized NeutrAvidin Products 1. Conti, L.R., et al. (2001). J. Biol. Chem. 276, 41270–41278.
2. Daniels, G.M. and Amara, S.G. (1998). Methods Enzymol. 296, 307–318.
Less nonspecific binding produces cleaner results and better yields. 3. Liaw, P.C.Y., et al. (2001). J. Biol. Chem. 276, 8364–8370.
4. Oda, Y., et al. (2001). Nature Biotechnology 19, 379–382.
When nonspecific binding is a problem in your application, 5. Hiller, Y., et al. (1987). Biochem. J. 248, 67–171.
6. Butler, J.E., et al. (1992). J. Immunol. Method 150, 77–90.
Thermo Scientific Immobilized NeutrAvidin Products are superior 7. Murakami, T., et al. (2000). Proc. Natl. Acad. Sci. USA 97(1), 343–348.
alternatives to avidin or streptavidin. NeutrAvidin Biotin-Binding 8. Cernuda-Morollon, E., et al. (2001). J. Biol. Chem. 276, 35530–35536.
9. Hiller, Y., et al. (1987). Biochem. J. 248, 67–171.
Protein is a modified avidin derivative that combines several 10. Kim, K., et al. (2001). J. Biol. Chem. 276, 40591–40598.
key features to provide biotin-binding with exceptionally low 11 Leighton, B.H., et al. (2002). J. Biol. Chem. 277, 29847–29855.
nonspecific binding properties. 12 Lesa, G.M., et al. (2000). J. Biol. Chem. 275, 2831–2836.
13. Trotti, D., et al. (2001). J. Biol. Chem. 276, 576–582.
Highlights:
• Carbohydrate-free – just like streptavidin, NeutrAvidin Biotin- Ordering Information
Binding Protein has no carbohydrate, eliminating nonspecific
binding problems due to sugars Product # Description Pkg. Size
• No interaction with cell surface molecules – absence of the 29200 NeutrAvidin Agarose Resin 5 ml
Arg-Tyr-Asp sequence (present in streptavidin), which mimics Support: Crosslinked 6% beaded agarose
Capacity: > 20 µg or 80 nmol biotin/ml resin
the universal cell surface recognition sequence present in a (approx. 1–2 mg biotinylated BSA/ml resin)
variety of molecules, eliminates cross-reactivity of cell surface
29201 NeutrAvidin Agarose Resin 10 ml
molecules Support and Capacity: Same as above
• Neutral pl – with a pl of 6.3, NeutrAvidin Protein has a pl that is 53150 NeutrAvidin UltraLink Resin 5 ml
closer to neutrality than avidin or streptavidin, eliminating Support: UltraLink Biosupport
electrostatic interaction that contributes to nonspecific binding Capacity: 12–20 µg biotin/ml gel
53151 NeutrAvidin Plus UltraLink Resin 5 ml

Applications: Capacity: ≥ 30 µg biotin/ml gel
• Immunoprecipitation 29202 High Capacity NeutrAvidin Agarose Resin 5 ml
• Purifying proteins that bind to biotinylated ligands Support: Crosslinked 6% beaded agarose
Capacity: > 75 µg biotin/ml resin
• Capturing biotinylated cell-surface proteins1-3 > 8 mg biotinylated BSA/ml resin
• Purifying biotinylated peptides4
29204 High Capacity NeutrAvidin Agarose Resin 10 ml
Immobilized Monomeric Avidin and Kit Immobilized Iminobiotin and Biotin

Ideal affinity support for gentle, reversible binding of biotinylated Iminobiotin offers mild dissociation conditions at pH 4.
proteins. NH
To break the avidin-biotin interaction, 8 M guanidine•HCl at pH 1.5 HN

NH
or boiling in SDS-PAGE sample buffer is required. These elution
methods may result in denaturation of the biotinylated protein and H H H
Agarose
cause irreversible damage to the support. In addition, avidin or N N N
Bead
S
streptavidin will be irreversibly denatured and lose the ability to
bind subsequent biotinylated samples. O
When avidin is coupled to a solid support as the subunit monomer,

Immobilized Iminobiotin
the specificity for biotin is retained, but the affinity for biotin bind-
ing substantially decreases (Ka ~ 108 M-1). The Monomeric Avidin
Agarose Resin and Kit can be used to bind biotinylated molecules, Iminobiotin is the guanido analog of biotin. The dissociation
and the bound material can be competitively eluted using 2 mM constant of the avidin-iminobiotin complex is pH-dependent.
biotin in phosphate-buffered saline (PBS). This technique provides At pH 9.5-11.0, the avidin-iminobiotin complex will bind tightly.
the gentlest elution conditions without contamination of the avidin At pH 4, the avidin-iminobiotin complex will dissociate. Because
subunits or substantial loss of column-binding capacity. denaturing agents such as 8 M guanidine•HCI or 4 M urea are
not used in the purification, an avidin conjugate has a better
Highlights:
chance of maintaining its activity during purification.
• Purifies biotinylated products under mild elution conditions
• Can be regenerated and reused at least 10 times Use immobilized D-Biotin as an “irreversible linkage” to bind
• Exhibits little nonspecific binding (3% or less) streptavidin conjugates. The biotin-streptavidin interaction can
withstand extremes in pH, salt and detergents.
References
Bernstein, E.M., et al. (1999). J. Biol. Chem. 274(2), 889–895. References
Sims, K.D., et al. (2000). J. Biol. Chem. 275(7), 5228–5237. Gitlin, G., et al. (1987). Biochem. J. 242, 923–926.
Ellerbroek, S.M., et al. (2001). J. Biol. Chem. 276, 24833–24842. Wood, G.S. and Warnke, R. (1981). J. Histochem. Cytochem. 29, 1196–1204.
Glover, B.P. and McHenry, C.S. (2001). Cell 105, 925–934. Hofmann, K., et al. (1980). Proc. Natl. Acad. Sci. USA 77(8), 4666–4668.
Horney, M.J., et al. (2001). J. Biol. Chem. 276, 2880–2889. Gao, C., et al. (1997). Proc. Natl. Acad. Sci. USA 94, 11777–11782.
Oda, Y., et al. (2001). Nature Biotechnology 19, 379–382. Hofmann, K., et al. (1980). Proc. Natl. Acad. Sci. USA 77, 4666–4668.
Schwarzman, A.L., et al. (1999). Proc. Natl. Acad. Sci. USA 96, 7932–7937.
Slatin, S.L., et al. (2002). Proc. Natl. Acad. Sci. USA 99, 1286–1291.
Product # Description Pkg. Size 20221 Iminobiotin Agarose Resin 5 ml
20228 Monomeric Avidin Agarose Resin 5 ml Spacer: Diaminodipropylamine
Support: Crosslinked 4% beaded agarose Capacity: ≥ 1 mg of avidin/ml resin
Capacity: ≥ 1.2 mg biotinylated BSA/ml resin
20218 Biotin Agarose Resin 5 ml
20267 Monomeric Avidin Agarose Resin 10 ml Support: Pierce CDI Support
Support and Capacity: Same as above Spacer: Diaminodipropylamine
Capacity: ≥ 2 mg of avidin/ml resin
20227 Monomeric Avidin Agarose Kit Kit
Includes: 1 x 2 ml Column, Binding and Elution buffers
53146 Immobilized Monomeric Avidin UltraLink Resin 5 ml

Capacity: ≥ 1.2 mg biotinylated BSA/ml resin
29129 Biotin 1g

Biotinylation reagent selection guide.
Membrane-
Product # Description Chemical Reactivity Water-Soluble Spacer Arm Length Cleavable Permeable†
21335* Sulfo-NHS-LC Biotin Primary Amine Yes 22.4 Å No No
21338 Sulfo-NHS-LC-LC-Biotin Primary Amine Yes 30.5 Å No No
21217* Sulfo-NHS-Biotin Primary Amine Yes 13.5 Å No No
21331* Sulfo-NHS-SS-Biotin Primary Amine Yes 24.3 Å Yes No
21442 NHS-SS-PEG4-Biotin Primary Amine Yes 37.9 Å Yes No
21362* NHS-PEG4-Biotin Primary Amine Yes 29 Å No No
21312* NHS-PEG12-Biotin Primary Amine Yes 56.0 Å No No
21303 TFA-PEG3-Biotin Primary Amine Yes 33.4 Å No No
21336 NHS-LC-Biotin Primary Amine No 22.4 Å No Yes
21343 NHS-LC-LC-Biotin Primary Amine No 30.5 Å No Yes
20217 NHS-Biotin Primary Amine No 13.5 Å No Yes
21441 NHS-SS-Biotin Primary Amine No 24.3 Å Yes Yes
21325* NHS-Chromogenic-Biotin Primary Amine No 41.0 Å No No
21117 NHS-Iminobiotin TFA Primary Amine No 13.5 Å No Yes
21218 PFP-Biotin Primary or Secondary Amine/ No 9.6 Å No Yes
RNA/DNA
21219 TFP-PEG3-Biotin Primary Amine Yes 32.6 Å No No
21901* Maleimide-PEG2-Biotin Sulfhydryl Yes 29.1 Å No No
21911 Maleimide-PEG11-Biotin Sulfhydryl Yes 59.1 Å No No
21900 Biotin-BMCC Sulfhydryl No 32.6 Å No Yes
21334 PEG-Iodoacetyl-Biotin Sulfhydryl Yes 24.7 Å No No
21333 Iodoacetyl-PEG2-Biotin Sulfhydryl No 27.1 Å No Yes
21341 Biotin-HPDP Sulfhydryl No 29.2 Å Yes Yes
21346 Amine-PEG2-Biotin Carboxyl‡ Yes 20.4 Å No No
21347 Amine-PEG3-Biotin Carboxyl ‡
Yes 22.9 Å No No
21345 Pentylamine-Biotin Carboxyl‡ Yes 18.9 Å No No
28020 Biocytin Hydrazide Carbohydrate/RNA/DNA Yes 19.7 Å No No
21339 Biotin Hydrazide Carbohydrate No 15.7 Å No Yes
21340 Biotin-LC-Hydrazide Carbohydrate No 24.7 Å No Yes
21360 Biotin-PEG4-Hydrazide Carbohydrate Yes 31.3 Å No No
29986 Psoralen-PEG3-Biotin DNA/RNA Protein Yes 36.9 Å No No
22020 PEG5-Biotin Dimer Avidin Cross-linking Yes 43.4 Å No No
29987 Photoactivatable Biotin DNA/RNA Protein No 30.0 Å No Yes
29982 Biotin-LC-ASA DNA/RNA Protein No 29.9 Å No Yes
28022 Biocytin Hydrazide Yes 20.1 Å No No
33033* Sulfo-SBED Trifunctional Yes N/A Yes No
33083 Mts-Atf-LC-Biotin Trifunctional Yes N/A Yes No
* Other product numbers (sizes and/or kits) also available; visit www.thermo.com/pierce for a complete listing.
† Membrane permeability is implied due to a molecule’s hydrophobic/hydrophilic nature.
‡ When used with EDC (Product # 22980, 22981).
NeutrAvidin Coated Polystyrene Plates Purified p60c-src Activity Detection with TK Peptide 2
1.5
The high affinity of avidin for biotin, without the nonspecific
Net Absorbance at 450 nm

binding problems.
Highlights: 1.0
• Easy and gentle immobilization of biotin-containing conjugates

• Lowest nonspecific binding properties of all biotin-binding proteins
0.5
• NeutrAvidin Biotin-Binding Protein has no carbohydrate and
an isoelectric point of 6.3
• No denaturing of the protein component of a conjugate upon 0
0.00 0.05 0.10 0.15
binding to the plate
Units Kinase
• Ideal for binding small hydrophilic molecules (e.g., peptides) that
typically exhibit poor binding directly to polystyrene
Biotinylated tyrosine kinase peptide 2 was added to Thermo Scientific
• Pre-blocked with your choice of Thermo Scientific Blocker™ BSA NeutrAvidin Coated Plates and incubated for 30 minutes. Wells were washed;
or SuperBlock® Blocking Buffer samples containing p60c-src tyrosine kinase were added to phosphorylate the
• Available in 96- and 384-well formats tyrosine residue on the peptide. Anti-phosphotyrosine monoclonal antibody
conjugated to horseradish peroxidase was added. Tyrosine kinase activity was
Characteristics of avidin-biotin proteins. detected by Thermo Scientific 1-Step™ Turbo TMB Substrate. Kinase activity
was quantitated by comparison with a standard curve generated using the
Isoelectric Contains Nonspecific phosphorylated form of the same peptide substrate.
Protein Point Carbohydrate Binding
Reference
Avidin 10–10.5 Yes High
Singh, Y., et al. (1999). Infect. Immun. 67, 1853–1859.
Streptavidin 5.5 No Low
NeutrAvidin 6.3 No Ultralow
Biotin-Binding Protein
Product # Coating Plate Type Blocking* Binding Capacity† Pkg. Size

15129 NeutrAvidin Protein, 100 µl Clear, 96-Well SuperBlock BB, 200 µl ~ 15 pmol biotin/well 5 plates
15127 NeutrAvidin Protein, 100 µl Clear, 8-Well Strip SuperBlock BB, 200 µl ~ 15 pmol biotin/well 5 plates
15116 NeutrAvidin Protein, 100 µl White, 96-Well SuperBlock BB, 200 µl ~ 15 pmol biotin/well 5 plates
15117 NeutrAvidin Protein, 100 µl Black, 96-Well SuperBlock BB, 200 µl ~ 15 pmol biotin/well 5 plates
15123 NeutrAvidin Protein, 200 µl Clear, 96-Well Blocker BSA, 300 µl > 15 pmol biotin/well 5 plates
15128 NeutrAvidin Protein, 200 µl Clear, 8-Well Strip Blocker BSA, 300 µl > 15 pmol biotin/well 5 plates
15216 NeutrAvidin Protein, 200 µl White, 96-Well Blocker BSA, 300 µl > 15 pmol biotin/well 5 plates
15217 NeutrAvidin Protein, 200 µl Black, 96-Well Blocker BSA, 300 µl > 15 pmol biotin/well 5 plates
15115 Pierce Biotin Binding Plate Sample Pack, one each of Product #s 15120, 15121, 15127, 15128 4 plates
* BB = Blocking Buffer
† Approximate values; plates tested for specific signal:noise and C.V.
All coated 96- and 384-well plates are available in bulk quantity with bulk packaging
at a discounted price.
We can also custom-coat plates using a certain type of plate or a specific supplier’s
plate or coat with a specific surface chemistry that is not included in our standard
product offering. Please contact our Large-Volume Custom Sales Team at 800-874-3723
or 815-968-0747 for more information. Outside the United States, contact your local branch
office or distributor.

10
NeutrAvidin High Binding Capacity (HBC)
HBC
Coated Plates 8 RBC
CHC
Unique technology for improved assay precision.
S/N Ratio
6
We offer researchers a wide variety of avidin-biotin products, 4

including our exclusive Thermo Scientific Pierce NeutrAvidin
Coated Plates available in a high binding capacity (HBC) format. 2
NeutrAvidin Protein is a deglycosylated form of avidin with a
near-neutral pI that results in less nonspecific binding than that of 0
0 5 10 15
streptavidin or avidin. Our patent-pending plate-coating technology
Biotinylated Phosphopeptide (pM/well)
offers a NeutrAvidin HBC Plate with a wider detection limit than
our regular binding capacity plates. The standard curve exhibits Comparison of Thermo Scientific NeutrAvidin High Binding Capacity (HBC)
greater linearity for detecting small biotinylated molecules such Coated Plate, NeutrAvidin Regular Binding Capacity (RBC) Coated Plates and
as peptides (see Figure) and oligonucleotides, resulting in greater another supplier’s Streptavidin Coated High Binding Capacity Plates (CHC).
assay precision. Try Thermo Scientific Pierce NeutrAvidin HBC Plates were incubated with various dilutions of biotinylated, phosphorylated
peptide. After washing, the plates were incubated with mouse anti-phospho-
Coated Plates for binding small biotinylated ligands and see the
tyrosine antibody (1:1,000) and then detected using an anti-mouse-FITC
difference. conjugate (1:666). The Y-axis is described as the signal-to-noise (S/N) ratio.
Highlights:
• Unique plate-coating technology – results in high loading of
NeutrAvidin Protein per well
• Improved sensitivity – less nonspecific binding for improved sig-
nal-to-noise ratios
• Broader dynamic range – extends the quantitative range so
there’s no need for dilutions
• Save time – pre-blocked plates to reduce the number of assay steps
• Flexible assay formats – coated plates offered in 96- and 384-well
formats and in different colors

15508 NeutrAvidin Protein, 100 µl Clear, 8-Well Strip SuperBlock BB, 200 µl ~ 60 pmol biotin/well 5 plates
Pierce Streptavidin Coated Polystyrene Plates

The specific binding affinity of streptavidin for biotin – in a microplate.
Highlights:
• Easy and gentle immobilization of biotin-containing conjugates
• Low nonspecific binding
• No denaturing of the protein component of a conjugate
upon binding
• Ideal for binding small biotinylated hydrophilic molecules
(e.g., peptides) that typically exhibit poor binding to polystyrene
• Pre-blocked with your choice of Blocker BSA or SuperBlock
Blocking Buffer
• Available in clear, white and black plates in 12 × 8-well strip,
96-well and 384-well formats
References
Estrada, G., et al. (1996). Mol. Cell Probes 10, 179–185.
Grobler, J.A. et al. (2002). Proc. Nat. Acad. Sci., USA 99, 6661–6666.

15124 Streptavidin, 100 µl Clear, 96-Well SuperBlock BB, 200 µl ~ 5 pmol biotin/well 5 plates
15126 Streptavidin, 100 µl Clear, 96-Well SuperBlock BB, 200 µl ~ 5 pmol biotin/well 5 x 5 plates
15120 Streptavidin, 100 µl Clear, 8-Well Strip SuperBlock BB, 200 µl ~ 5 pmol biotin/well 5 plates
15122 Streptavidin, 100 µl Clear, 8-Well Strip SuperBlock BB, 200 µl ~ 5 pmol biotin/well 5 x 5 plates
15118 Streptavidin, 100 µl White, 96-Well SuperBlock BB, 200 µl ~ 5 pmol biotin/well 5 plates
15119 Streptavidin, 100 µl Black, 96-Well SuperBlock BB, 200 µl ~ 5 pmol biotin/well 5 plates
15125 Streptavidin, 200 µl Clear, 96-Well Blocker BSA, 300 µl ~ 10 pmol biotin/well 5 plates
15121 Streptavidin, 200 µl Clear, 8-Well Strip Blocker BSA, 300 µl ~ 10 pmol biotin/well 5 plates
15218 Streptavidin, 200 µl White, 96-Well Blocker BSA, 300 µl ~ 10 pmol biotin/well 5 plates
15219 Streptavidin, 200 µl Black, 96-Well Blocker BSA, 300 µl ~ 10 pmol biotin/well 5 plates
15115 Pierce Biotin Binding Plate Sample Pack, one each of Product #s 15120, 15121, 15127, 15128 4 plates

Pierce Streptavidin HBC Coated Plates
180
Take advantage of our technology that provides a broader
160 HBC
dynamic range.
140 CHC
Thermo Scientific Pierce Streptavidin High Binding Capacity 120
S/N Ratio
(HBC) Coated Plates are designed for binding biotinylated 100
oligonucleotides and peptides with higher binding efficiency 80

than other commercially available plates. Our proprietary coating 60
technology (patent pending) has created a streptavidin-coated 40
plate with four- to five-times the binding capacity of other suppliers’ 20
plates. Using our Streptavidin HBC Plate can result in an assay 0
0 5 10 15 20 25 30
with a broader dynamic range and better linearity, leading to
Fluoresceinated Oligonucleotide (pM/well)
improved assay precision (see Figure). Try our Streptavidin
HBC Coated Plate and see what has been going undetected in
your research. Comparison of Thermo Scientific Pierce Streptavidin High Binding Capacity
(HBC) Coated Plate with another commercially available high binding capacity
Highlights: plate (CHC). Plates were incubated with a biotinylated oligonucleotide, washed
and probed with a complementary oligonucleotide labeled with fluorescein at
• Broader dynamic range – extends the quantitative range so various dilutions. The Y-axis is described as the signal-to-noise (S/N) ratio.
there’s no need for dilutions
• Better sensitivity – increased binding capacity allows direct
detection of small ligands not observed with regular binding
capacity plates
• Superior assay precision – standard curve demonstrates
greater linearity
• Save time – pre-blocked to reduce number of assay steps
• Flexible assay formats – offered in 96- and 384-well formats
and in different colors

15501 Streptavidin, 100 µl Clear, 8-Well Strip SuperBlock BB, 200 µl ~ 125 pmol biotin/well 5 plates
Affinity Purification of Antibodies
General Purification of Immunoglobulins

Because antibodies have predictable structure, including relatively
invariant domains, it has been possible to identify certain protein
ligands that are capable of binding generally to antibodies, regard-
less of the antibody’s specificity to antigen. Protein A, Protein G
and Protein L are three bacterial proteins whose antibody-binding
properties have been well characterized. These proteins have
been produced recombinantly and used routinely for affinity
purification of key antibody types from a variety of species. A
genetically engineered recombinant form of Protein A and G,
called Protein A/G, is also available. These antibody-binding
proteins are available immobilized to beaded agarose resin,
UltraLink Biosupport and coated onto microplates (see previous
section on Solid Supports for Affinity Purification, page 6).
Proteins A, G, A/G and L bind to antibodies at sites other than the

antigen-binding domain. Therefore, these proteins can be used in
purification schemes such as immunoprecipitation (see discussion
Antibodies specific for an antigen of interest are one of the of Immunoprecipitation that follows, page 54).
most useful and important tools that biology researchers can
possess. The production and use of specific antibodies as Proteins A, G, A/G and L have unique properties, which make each
detection probes and purification ligands (i.e., immunotechnology) one suitable for different types of antibody targets (e.g., antibody
has revolutionized bioresearch and diagnostic technologies. subclass or animal species). It is important to realize that use of
Animals immunized with prepared antigens will produce Protein A, G or L results in purification of general immunoglobulin
specific antibodies against the antigen. When purified from from a crude sample. Depending on the sample source, antigen-
serum or hybridoma cell lines that are prepared from tissue of specific antibody may account for only a small portion of the total
the immunized animal, the antibody can be used directly (or immunoglobulin in the sample. For example, generally only 2-5%
after labeling with enzyme or fluorescent tags) to probe the of total IgG in mouse serum is specific for the antigen used to
specific antigen in Western blotting, ELISA or a variety of other immunize the animal.
applications. Antibodies are most commonly purified by one of
two affinity purification methods: general immunoglobulin
purification or specific antibody purification. Immobilized Protein L, Protein A, Protein G
and Protein A/G
We offer these popular antibody-binding proteins immobilized on
several different resins, beads and plates for use in immunoaffinity
purification techniques. All four proteins (A, G, A/G and L) are
available as purified recombinants immobilized to crosslinked 6%
beaded agarose. This is the traditional format historically used for
small-scale column purification and immunoprecipitation methods.
Our agarose resins differ from those typically offered by other
suppliers in that our immobilization method is more stable and
results in less nonspecific binding. We also offer “Plus” versions
of the Protein A, G, A/G and L agarose resins, which contain twice
the amount of protein per milliliter of resin and provide for nearly
twice the antibody binding capacity.
Protein A, G, A/G and L are also available immobilized to UltraLink

Biosupport, an extremely durable, polyacrylamide-based resin
with very low nonspecific binding characteristics. The UltraLink
Format is a perfect support for working with large volume samples
in large-scale purification methods requiring fast flow and high
pressure.
The interaction between the various proteins and IgG is not

equivalent for all species or all antibody subclasses. The table
on the following page will help you decide which affinity protein
is best for your application.

Characteristics of immunoglobulin-binding proteins.
Recombinant Native Recombinant Recombinant Recombinant

Protein L Protein A Protein A Protein G Protein A/G
Production Source E. coli S. aureus E. coli E. coli E. coli
Molecular Weight 35,800 46,700 44,600 21,600 50,460
Number of Binding Sites for IgG 4 4 4 2 6
Albumin-Binding Site No No No No No
Optimal Binding pH 7.5 8.2 8.2 5 5–8.2
Binds to VLK FC FC FC FC
Thiophilic Thiophilic
Protein A Protein G Protein A/G Protein L† Adsorbent Protein A Protein G Protein A/G Protein L† Adsorbent
Human IgG s s s s m Human IgG2 s s s s m
Mouse IgG s s s s s Human IgG3 w s s s m
Rabbit IgG s s s w m Human IgG4 s s s s m
Goat IgG w s s nb s Human Fab w w w s m
Rat IgG w m m s s Human ScFv w nb w s m
Sheep IgG w s s nb s Mouse IgG1 w m m s s
Cow IgG w s s nb s Mouse IgG2a s s s s s
Guinea Pig IgG s w s – s Mouse IgG2b s s s s s
Hamster IgG m m m s – Mouse IgG3 s s s s s
Pig IgG s w s s s Rat IgG1 w m m s s
Horse IgG w s s – s Rat IgG2a nb s s s s
Donkey IgG m s s – – Rat IgG2b nb w w s s
Dog IgG s w s – s Rat IgG2c s s s s s
Cat IgG s w s – s Cow IgG1 w s s nb s
Monkey IgG s s s – s Cow IgG2 s s s nb s
(Rhesus)
Sheep IgG1 w s s nb s
Chicken IgY nb nb nb nb m
Sheep IgG2 s s s nb s
Human IgM w nb w s m
Goat IgG1 w s s nb s
Human IgE m nb m s –
Goat IgG2 s s s nb s
Human IgD nb nb nb s –
Horse IgG(ab) w nb w – s
Human IgA w nb w s m
Horse IgG(c) w nb w – s
Human IgA1 w nb w s m
Horse IgG(T) nb s s – s
Human IgA2 w nb w s m
Mouse IgM nb nb nb s m
Human IgG1 s s s s m
w = weak binding, m = medium binding, s = strong binding, nb = no binding, – means information not available
* Data represent a summary of binding properties reported in the literature. Inevitably some discrepancies exist among reported values as a result of differences in binding
buffer conditions and form of the proteins used.
† Binding will occur only if the appropriate kappa light chains are present. Antibodies lambda light chains will not bind, regardless of their class and subclass.
Affinity Purification of Antibodies
Protein A of purification. Supplied as a 50% resin slurry in storage buffer,

Immobilized Protein A Agarose is the usual choice either for
Protein A Characteristics and IgG Binding Properties small-scale batch method purification procedures or for packing
Protein A is a cell wall component produced by several strains of gravity-flow columns.
Staphylococcus aureus. It consists of a single polypeptide chain
(MW 46.7 kDa) and contains little or no carbohydrate.1 Protein A Our Immobilized Protein A is also available on Trisacryl® GF-2000,
binds specifically to the Fc region of immunoglobulin molecules, rather than agarose resin. This stable affinity support can withstand
especially IgG. It has four high-affinity (Ka = 108 M-1) binding sites the high-throughput volumes required in large-scale purification
that are capable of interacting with the Fc region of IgGs of several procedures. In addition, because Trisacryl GF-2000 is a hydrophilic
species.2 The molecule is heat-stable and retains its native confor- matrix, nonspecific binding of proteins is minimized.
mation even after exposure to denaturing reagents such
as 4 M urea, 4 M thiocyanate and 6 M guanidine hydrochloride.3 Thermo Scientific Pierce Protein A UltraLink Resin is another
alternative for large-scale, high-throughput applications. UltraLink
In its immobilized form (e.g., covalently coupled to beaded
Biosupport is composed of a hydrophilic, crosslinked bis-
agarose resin), Protein A has been used extensively for isolation
acrylamide/azlactone copolymer. It has an average bead diameter
of a wide variety of immunoglobulins from several species of
of 60 µm, can withstand pressures exceeding 100 psi and retains
mammals. However, the interaction between Protein A and IgG
good chromatographic properties using flow rates up to 3,000
is not equivalent for all animal sources and subclasses of IgG.
cm/hour. Our Protein A UltraLink Resin is the ideal choice for
For example, human IgG1, IgG2 and IgG4 bind strongly to Protein A,
medium pressure liquid chromatographic systems.
while IgG3 does not bind.2 In mice, IgG2a , IgG2b and IgG3 bind
strongly to Protein A, but IgG1 (the dominant subclass in serum) Thermo Scientific Pierce Recombinant Protein A Agarose (Product
binds only weakly using standard buffer conditions. Most rat IgG #s 20365 and 20366) uses a genetically engineered form of Protein
subclasses bind weakly or not at all to Protein A. Despite this A that is produced recombinantly in a nonpathogenic form of
variability, Protein A is very effective for routine affinity purification Bacillus. Non-essential regions have been removed, and five
of IgG from the serum of many species. It is especially suited for IgG-binding sites are included, resulting in a mass of 44.6 kDa. Some
purification of polyclonal antibodies from rabbits. researchers believe that the recombinant form should be used if
the antibody preparation has strict requirements for being
Weak binding of Protein A to mouse IgG1 using traditional Tris•HCl
enterotoxin-free. Otherwise, the native form serves as a highly
or sodium phosphate buffer systems is of particular concern and
efficient means for purifying antibodies. Our Recombinant
is one reason to choose Protein G when purifying mouse antibodies.
Protein A Agarose is also compatible with Pierce Binding and
However, we have developed a binding buffer that allows Protein A
Elution Buffers.
to bind mouse IgG1 nearly as well as other subclasses (see subse-
quent discussion of IgG Binding and Elution Buffers, page 44). Thermo Scientific NAb™ Protein A Spin Columns are available in
three package/column sizes: 10 x 0.2 ml Protein A resin in a 1 ml
The variable binding properties of Protein A for different subclasses
spin column, 5 x 1 ml resin in a 5 ml spin column, and 1 x 5 ml resin
of IgG can be used advantageously to separate one IgG type from
in a 22 ml spin column. For the greatest convenience, choose
another. Antibodies that do not bind to immobilized Protein A
NAb Protein A Plus Spin Kits (Product #s 89948 and 89978), which
can be recovered by collecting the non-bound (“flow-through”)
include pre-packed columns of our Protein A Plus Agarose, as well
fractions during binding and wash steps in an affinity purification
as binding, elution and neutralization buffers. These kits contain
procedure. In this way, human IgG3 and other immunoglobulin
everything needed to isolate IgG from serum, ascites or cell cul-
subclasses can be isolated from those that do bind to Protein A;
ture supernatants through a fast and simple process that requires
however, other IgGs and serum proteins, such as albumin, will also
approximately 30 minutes to complete. The columns in the NAb
be present in the non-bound fraction. Certain IgM, IgD and IgA
Kits can each be regenerated a minimum of 10 times without a sig-
molecules also do not bind to Protein A and can be separated from
nificant loss of binding capacity. NAb Protein A Spin Kits are avail-
Protein A-binding proteins in the same manner.
able in two sizes: 10 x 0.2 ml spin column kit and 2 x 1 ml spin col-
Immobilized Protein A Products umn kit. The spin format of these columns and kits greatly reduces
Thermo Scientific Pierce Immobilized Protein A is offered on several the time required to process serum, ascites and cell
different solid supports and made available in different binding culture supernatants and produce a purified antibody preparation.
capacity formats, package sizes and kit formats. Protein A Agarose The NAb Columns and Kits are also compatible with gravity-flow
generally denotes products composed of highly purified Protein A and vacuum-based purification methods.
that is covalently coupled to crosslinked 6% beaded agarose resin.
The Thermo Scientific Pierce Protein A Chromatography Cartridges
Our Protein A Agarose is available with different densities of (Product #s 89924 and 89925) are designed for fast, consistent sep-
Protein A ligand bound to the resin. The standard version has arations using a syringe, pump or chromatography system. They
a binding capacity of 12–19 mg of human IgG per ml of resin, while are available in 1 ml and 5 ml sizes. The column inlet and outlet are
the plus version has a binding capacity of > 35 mg of human IgG molded with 1/16” threads, and adaptors are included for coupling
per ml of resin. Both resins exhibit excellent elution properties directly to most chromatography systems. Luer-Lok Adaptors are
when used with Pierce Buffer Systems (Figure 1), which generally also provided with the columns for simple attachment to a syringe.
enable the resin to be regenerated and used for at least 10 rounds

mAU FT Thermo Scientific Immobilized Protein A
E
3000 Plus Products
Twice the amount of coupled Protein A per milliliter of resin.
Absorbance at 280 nm
2500
2000
1500
1000
22810 Protein A Plus Agarose 1 ml
500
Capacity: > 35 mg human IgG/ml resin;
16–17 mg mouse IgG/ml resin
0
0.0 5.0 10.0 15.0 20.0 25.0 30.0 35.0 ml 22811 Protein A Plus Agarose 5 ml
Affinity chromatographic purification of mouse IgG from mouse ascites fluid 22812 Protein A Plus Agarose 25 ml
using Thermo Scientific Pierce Protein A Agarose and the IgG Binding and Support and Capacity: Same as above
Elution Buffer System. From 1 ml of mouse ascites fluid, 5.5 mg of mouse IgG
89924 Pierce Chromatography Cartridges, Protein A 2 x 1 ml
was recovered. Support and Capacity: Same as above
Thermo Scientific MagnaBind Protein A (Product # 21348) is 89925 Pierce Chromatography Cartridge, Protein A 1 x 5 ml
available to perform benchtop magnetic separations quickly
and easily. MagnaBind Beads consist of a silanized surface 89952 NAb Protein A Plus Spin Columns 10 x 0.2 ml
over a core of superparamagnetic iron oxide. Protein A has
been attached to these beads to allow IgG removal, IgG 89956 NAb Protein A Plus Spin Columns 5 x 1 ml
purification or magnetic immunoprecipitation.
89960 NAb Protein A Plus Spin Column 1 x 5 ml
References Support and Capacity: Same as above
1. Sjoquist, J., et al. (1972). Eur. J. Biochem. 29, 572–578.
2. Hjelm, H., et al. (1975). Eur. J. Biochem. 57, 395–403. 89948 NAb Protein A Plus Spin Purification Kit Kit
3. Sjoholm, I., et al. (1975). Eur. J. Biochem. 51, 55–61. Includes: Protein A Spin Columns 10 x 0.2 ml
PBS Binding Buffer 500 ml
Neutralization Buffer 7 ml
Thermo Scientific Immobilized Protein A Products Collection Tubes 10 x 0.2 ml
89978 NAb Protein A Plus Spin Purification Kit Kit

Ordering Information Includes: Protein A Spin Columns 2 x 1 ml
Product # Description Pkg. Size Neutralization Buffer 7 ml
20333 Protein A Agarose 5 ml 53142 Protein A UltraLink Plus Resin
5 ml
Capacity: 12–19 mg human IgG/ml resin
Capacity: > 30 mg human IgG/ml resin
20334 Protein A Agarose 25 ml 45202 Protein A Spin Plate
1 plate
96–well filter plate containing Protein A Agarose
20356 Protein A Columns 5 x 1 ml for IgG screening
44667 Protein A IgG Purification Kit Kit

Includes: Protein A Columns 5 x 1 ml
Protein A IgG Binding Buffer 1,000 ml
Desalting Columns 5 x 5 ml
20338 Protein A Trisacryl Resin 5 ml

Support: Trisacryl GF 2000
53139 Protein A UltraLink Resin 5 ml

Capacity: > 16 mg of human IgG/ml resin
Thermo Scientific Products for Affinity Purification of Antibodies
MagnaBind Protein A Beads Protein G
Ordering Information Protein G Characteristics and IgG Binding Properties

Protein G is a bacterial cell wall protein isolated from group
G streptococci.1 Like Protein A from Staphylococcus aureus,
Protein G binds to most mammalian immunoglobulins primarily
21348 MagnaBind Protein A Beads 5 ml through their Fc regions. Protein G binds weakly to Fab fragments.1
Support: 1–4 µm iron oxide particles
Capacity: > 0.2 mg rabbit IgG/ml beads Sequencing of DNA that encodes native Protein G indicates that
there are two immunoglobulin binding sites, as well as albumin
and cell surface binding sites.2 In the recombinant form of
Protein A Coated Microplates Protein G, these albumin and cell surface binding sites have
been eliminated to reduce nonspecific binding when purifying
Ordering Information immunoglobulins. With the albumin site removed, recombinant
Protein G can be used to separate albumin from crude human
Product # Description Pkg. Size immunoglobulin samples. Recombinant Protein G has a mass of
15130 Protein A, Clear 96-Well Plates 5 plates approximately 22 kDa. However, its apparent mass by SDS-PAGE
Coating Volume: 100 µl is nearly 34 kDa.
Blocking: SuperBlock Blocking Buffer, 200 µl
Capacity: ∼ 4 pmol rabbit IgG/well
Immobilized Protein G is most commonly used for the purification
15132 Protein A, Clear 8-Well Strip Plates 5 plates of mammalian monoclonal and polyclonal antibodies that do not
Specifications: Same as above bind well to Protein A. It has been reported that most mammalian
15154 Protein A, White 96-Well Plates 5 plates immunoglobulins bind with greater affinity to Protein G than
Specifications: Same as above Protein A.1 There are, however, species to which Protein A has
15155 Protein A, Black 96-Well Plates 5 plates greater affinity.3 Protein G binds with significantly greater affinity
Specifications: Same as above to several immunoglobulin subclasses including human IgG3 and
rat IgG2a. Unlike Protein A, Protein G does not bind to human IgM,
Recombinant Protein A Agarose IgD or IgA.1
Our recombinant form of immobilized Protein A, manufactured Differences in binding characteristics between Protein A and
with a leak-resistant linkage. Protein G are explained by differences in the immunoglobulin
binding sites of each protein. Although the tertiary structures
References of these proteins are similar, their amino acid compositions
1. Bjork, I., et al. (1972). Eur. J. Biochem. 29, 579–584.
2. Goding, J.W. (1978). J. Immunol. Method 20, 241–253. differ significantly.
3. Lindmark, R., et al. (1983). J. Immunol. Method 62, 1–13.
4. Surolia, A., et al. (1982). Trends Biochem. Sci. 7, 74–76. Inconsistency in reporting of Protein G binding characteristics
5. Kronvall, G., et al. (1970). J. Immunol. 105,1116–1123.
6. Reeves, H.C., et al. (1981). Anal. Biochem. 115 194–196.
occurs in the literature. One cause for this inconsistency likely
7. Kilion, J.J. and Holtgrewe, E.M. (1983). Clin. Chem. 29, 1982–1984. results from differences in the particular source and isolation
8. Ey, P.L., et al. (1978). Immunochemistry 15, 429–436. method used for the native Protein G characterized in each study.
9. Bigbee, W.L., et al. (1983). Mol. Immunol. 20, 1353–1362.
In addition, several methods have been used to assess relative
binding affinity including radiolabeling experiments and ELISA
Ordering Information techniques, the results of which are not directly comparable.
Finally, significant binding differences result from different
binding buffers used with Protein G. Optimal binding for most
immunoglobulins to Protein G occurs in sodium acetate buffer,
20365 Recombinant Protein A Agarose 5 ml pH 5.0,4 although many studies have used more neutral Tris or
Support: Crosslinked 6% beaded agarose resin
Capacity: ≥ 12 mg human IgG/ml resin using the phosphate buffers for binding. Approximately 44% more IgG from
IgG Buffer System rat serum bound to Protein G using acetate buffer, pH 5.0 (e.g.,
20366 Recombinant Protein A Agarose 25 ml Protein G IgG Binding Buffer, Product # 21011) compared
Support and Capacity: Same as above to Tris•HCl pH 7.5 buffer.
Immobilized Protein G Products

Like Immobilized Protein A already discussed, Thermo Scientific
Pierce Immobilized Protein G is offered in several package sizes,
columns and kit formats for your convenience in gravity-flow,
spin and automated purification procedures. Our Immobilized
Protein G products incorporate the recombinant form of Protein G
immobilized to either crosslinked 6% beaded agarose or UltraLink
Biosupport. For a more detailed description of supports, see the
previous page about Pierce Immobilized Protein A Products. Both
types of Immobilized Protein G use coupling chemistries that are

leak-resistant and provide a matrix with minimal nonspecific binding. Ordering Information
Both supports can be regenerated and reused multiple times when
stored properly. 53125 Protein G UltraLink Resin 2 ml
Capacity: > 20 mg of human IgG/ml resin
The new Thermo Scientific Pierce Protein G Chromatography
53126 Protein G UltraLink Resin 10 ml
Cartridges (Product #s 89926 and 89927) are designed for fast, Support and Capacity: Same as above
consistent separations using a syringe, pump or chromatography
system. They are available in 1 ml and 5 ml sizes. Column inlet and 53127 Protein G UltraLink Columns 2 x 2 ml
outlet are molded with 1/16” threads and adaptors are included
for coupling directly to most chromatography systems. Luer-Lok 45204 Protein G Spin Plate 1 plate
96-well filter plate containing Protein G Agarose
Adaptors are also provided with the columns for simple attachment for IgG screening
to a syringe.
For the greatest convenience, choose Thermo Scientific NAb Immobilized Protein G Plus Products
Protein G Spin Kits (Product #s 89949 and 89979), which include
pre-packed columns of our Protein G Agarose, as well as binding, Twice the amount of coupled Protein G per milliliter of resin.
elution and neutralization buffers. These kits contain everything
needed to isolate IgG from serum, ascites or cell culture superna- Ordering Information
tants through a fast and simple process that requires approximate-
ly 30 minutes to complete. The columns in the NAb Kits can each Product # Description Pkg. Size
be regenerated a minimum of 10 times without a 22851 Protein G Plus Agarose 2 ml
significant loss of binding capacity. Support: Crosslinked 6% beaded agarose
References
1. Bjorck, L. and Kronvall, G. (1984). J. Immunol. 133, 969–974.
22852 Protein G Plus Agarose 10 ml
2. Guss, B., et al. (1986). EMBO J. 5, 1567–1575.
3. Eliasson, M., et al. (1988). J. Biol. Chem. 263, 4323–4327. 53128 Protein G Plus UltraLink Resin 2 ml
4. Åkerström, B. and Bjorck, L. (1986). J. Biol. Chem. 261, 10240–10247. Support: UltraLink Biosupport
Immobilized Protein G Products
MagnaBind Protein G Beads
Product # Description Pkg. Size Ordering Information

20398 Protein G Agarose 2 ml
Support: Crosslinked 6% beaded agarose Product # Description Pkg. Size
Capacity: 11–15 mg human IgG/ml resin 21349 MagnaBind Protein G Beads 5 ml
20399 Protein G Agarose Support: 1–4 µm iron oxide particles
10 ml
Support and Capacity: Same as above Capacity: > 0.2 mg rabbit IgG/ml beads
20397 Protein G Agarose 25 ml

Support and Capacity: Same as above Protein G Coated Microplates
89926 Pierce Chromatography Cartridges, Protein G 2 x 1 ml
89927 Pierce Chromatography Cartridge, Protein G 1 x 5 ml
89953 NAb Protein G Spin Columns 10 x 0.2 ml
15131 Protein G, Clear 96-Well Plates 5 plates
Coating Volume: 100 µl
89957 NAb Protein G Spin Columns 5 x 1 ml Blocking: SuperBlock Blocking Buffer, 200 μl
Support and Capacity: Same as above Capacity: ∼ 2 pmol rabbit IgG/well
89961 NAb Protein G Spin Columns 1 x 5 ml 15133 Protein G, Clear 8-Well Strip Plates 5 plates
Support and Capacity: Same as above Specifications: Same as above
89949 Nab Protein G Spin Purification Kit Kit 15156 Protein G, White 96-Well Plates 5 plates
Includes: Protein G Spin Columns 10 x 0.2 ml Specifications: Same as above
IgG Elution Buffer
15157 Protein G, Black 96-Well Plates 5 plates
50 ml
Specifications: Same as above
Collection Tubes 10 x 2 ml
89979 Nab Protein G Spin Purification Kit Kit

Includes: Protein G Spin Columns 2 x 1 ml
Protein A/G Immobilized Protein A/G Products

Protein A/G is a genetically engineered protein that combines the Ordering Information
IgG binding profiles of both Protein A and Protein G. Protein A/G
is a gene fusion product with a mass of 50.5 kDa, designed to
contain four Fc binding domains from Protein A and two from
Protein G. Protein A/G is not as pH-dependent as Protein A 20421 Protein A/G Agarose 3 ml
(see Figure on page 45) but otherwise has the additive properties Capacity: > 7 mg human IgG/ml resin
of Protein A and G.
20422 Protein A/G Agarose 15 ml
Protein A/G binds to all human IgG subclasses. In addition, it
binds to IgA, IgE, IgM and, to a lesser extent, IgD. Protein A/G 89930 Pierce Chromatography Cartridges, Protein A/G 2 x 1 ml
also binds well to all mouse IgG subclasses but does not bind
mouse IgA, IgM or serum albumin.1 This makes Protein A/G an 89931 Pierce Chromatography Cartridge, Protein A/G 1 x 5 ml
excellent tool for purification and detection of mouse monoclonal
antibodies from IgG subclasses, without interference from IgA, 89954 NAb Protein A/G Spin Columns 10 x 0.2 ml
IgM and murine serum albumin. Individual subclasses of mouse
monoclonals are more likely to have a stronger affinity to the 89958 NAb Protein A/G Spin Columns 5 x 1 ml
chimeric Protein A/G than to either Protein A or Protein G.2
89962 NAb Protein A/G Spin Column 1 x 5 ml
Immobilized Protein A/G is an ideal choice for purification of Support and Capacity: Same as above
polyclonal or monoclonal IgG antibodies whose subclasses have 89950 NAb Protein A/G Spin Purification Kit Kit
not been determined. Overall binding capacity is greater when Includes: Protein A/G Spin Columns 10 x 0.2 ml
pH 8.0 buffer (optimal for Protein A) is used rather than pH 5.0 IgG Elution Buffer 50 ml
buffer, which is optimal for Protein G used alone. Furthermore, Neutralization Buffer 7 ml
Collection Tubes 10 x 2 m
Thermo Scientific Pierce Protein A Binding Buffer provides for
greater binding than Tris•HCl, pH 8.0 (see description of IgG 89980 NAb Protein A/G Spin Purification Kit Kit
Binding and Elution Buffers on page 45). Includes: Protein A/G Spin Columns 2 x 1 ml
Immobilized Protein A/G Products Neutralization Buffer 7 ml
Like Immobilized Protein A and G already discussed, Thermo
53132 Protein A/G UltraLink Resin 2 ml
Scientific Pierce Immobilized Protein A/G is offered in several Support: UltraLink Biosupport
package sizes, columns and kit formats for your convenience Capacity: > 20 mg human IgG/ml resin
in gravity-flow, spin and automated purification procedures. 53133 Protein A/G UltraLink Resin 10 ml
The new Thermo Scientific Pierce Protein A/G Chromatography
Cartridges (Product # 89930 and 89931) are designed for fast,
consistent separations using a syringe, pump or chromatography Immobilized Protein A/G Plus Products
system. They are available in 1 ml and 5 ml sizes. Column inlet and
Twice the amount of coupled Protein A/G per milliliter of resin.
outlet are molded with 1/16” threads and adaptors are included
for coupling directly to most chromatography systems. Luer-Lok
Adaptors are also provided with the columns for simple Ordering Information
attachment to a syringe.
For the greatest convenience, choose Thermo Scientific 20423 Protein A/G Plus Agarose 2 ml
NAb Protein A/G Spin Kits (Product #s 89950 and 89980) which Support: Crosslinked 6% beaded agarose
include pre-packed columns of Protein A/G Agarose, as well as
binding, elution and neutralization buffers. These kits contain 53135 Protein A/G Plus on UltraLink Support 2 ml
everything needed to isolate IgG from serum, ascites or cell Capacity: > 28 mg human IgG/ml resin
culture supernatants through a fast and simple process that
requires approximately 30 minutes to complete. The columns in
the NAb Kits can each be regenerated a minimum of 10 times Protein A/G Coated Microplates
without a significant loss of binding capacity.
Reference
1. Eliasson, M., et al. (1988). J. Biol. Chem. 263, 4323–4327
15138 Protein A/G, Clear 96-Well Plates 5 plates
Blocking: SuperBlock Blocking Buffer, 200 µl
Capacity: ∼ 5 pmol rabbit IgG/well

Protein L Immobilized Protein L Products
Protein L is an immunoglobulin-binding protein (35.8 kDa) that Ordering Information
originates from the bacteria Peptostreptococcus magnus, but is
now produced recombinantly. Unlike Protein A and Protein G,
which bind primarily through Fc regions (i.e., heavy chain) of
immunoglobilins, Protein L binds immunoglobulins through interactions 20510 Protein L Agarose 2 ml
with their light chains. Since no part of the heavy chain is involved Capacity: 5–10 mg human IgG/ml resin
in the binding interaction, Protein L binds a wider range of Ig classes than
20512 Protein L Agarose 10 ml
Protein A or G. Protein L will bind to representatives of all classes of Ig Support and Capacity: Same as above
including IgG, IgM, IgA, IgE and IgD. Single-chain variable fragments
89928 Pierce Chromatography Cartridges, Protein L 2 x 1 ml
(ScFv) and Fab fragments can also be bound by Protein L. Support and Capacity: Same as above
Despite this wide-ranging binding capability with respect to Ig classes 89929 Pierce Chromatography Cartridge, Protein L 1 x 5 ml
(which are defined by heavy chain type), Protein L is not a universal
immunoglobilin-binding protein. Binding of Protein L to immunoglobu- 89955 NAb Protein L Spin Columns 10 x 0.2 ml
lins is restricted to those containing kappa light chains (i.e., κ chain of
the VL domain).1 In humans and mice, kappa (κ) light chains 89959 NAb Protein L Spin Columns 5 x 1 ml
predominate. The remaining immunoglobulins have lambda (λ) light
chains. Furthermore, Protein L is effective in binding only certain 89963 NAb Protein L Spin Column 1 x 5 ml
subtypes of kappa light chains. For example, it binds human VκI, VκIII
and VκIV subtypes but does not bind the VκII subtype. Binding of mouse 89951 NAb Protein L Spin Purification Kit Kit
Includes: Protein L Spin Columns 10 x 0.2 ml
immunoglobulins is restricted to those having VκI light chains.1 PBS Binding Buffer 500 ml
Given these specific requirements for effective binding, immobilized Neutralization Buffer 7 ml
Collection Tubes 10 x 2 m
Protein L is not appropriate for general polyclonal antibody purification
from serum, which contains a mixture of immunoglobulins having 89981 NAb Protein L Spin Purification Kit Kit
different types of light chains. The main application for immobilized Includes: Protein L Spin Columns 2 x 1 ml
Protein L is purification of monoclonal antibodies from ascites or IgG Elution Buffer 240 ml
culture supernatant that are known to have the VκI light chain. Neutralization Buffer 7 ml
Protein L is extremely useful for this specific application because
it does not bind bovine immunoglobilins, which are present in the
Immobilized Protein L Plus Products
media serum supplement. Also, in contrast to Protein A and G,
Protein L is very effective at binding IgM. Although it binds to the Twice the amount of coupled Protein L per milliliter of resin.
Fab portion of the immunoglobulin monomer, Protein L does not
interfere with the antigen-binding site of the antibody. Therefore, Ordering Information
Protein L potentially can be used in immunoprecipitation (IP)
procedures.
Immobilized Protein L Products 20520 Protein L Plus Agarose 2 ml
Like Immobilized Protein A and G already discussed, Thermo Support: Crosslinked 6% beaded agarose
Capacity: > 10–20 mg human IgG/ml resin
Scientific Pierce Immobilized Protein L is offered in several
package sizes, columns and kit formats for your convenience in
gravity-flow, spin and automated purification procedures. Protein L Coated Microplates
The new Thermo Scientific Pierce Protein L Chromatography Ordering Information
Cartridges (Product #s 89928 and 89929) are designed for fast,
consistent separations using a syringe, pump or chromatography Product # Description Pkg. Size
system. They are available in 1 ml and 5 ml sizes. Column inlet
and outlet are molded with 1/16” threads and adaptors are includ- 15190 Protein L, Clear 96-Well Plates 5 plates
ed for coupling directly to most chromatography systems. Luer-Lok Blocking: SuperBlock Blocking Buffer, 200 µl
Adaptors are also provided with the columns for simple attach-
ment to a syringe.
For the greatest convenience, choose Thermo Scientific NAb Protein L

Spin Kits (Product #s 89951 and 89981) which include pre-packed
columns of Protein L Agarose, as well as binding, elution and
neutralization buffers.
Reference
1. Nilson, B., et al. (1992). J. Biol. Chem. 267, 2234–2238.
IgG Binding and Elution Buffers for The Thermo Scientific Pierce Protein A IgG Binding Buffer is a
unique, phosphate-based formulation (pH 8.0) that achieves maxi-
Protein A, G, A/G and L mum binding capacity of IgG to immobilized Protein A. Overall IgG
Binding and Elution Steps in Affinity Purification binding capacity is increased with this buffer relative to traditional
binding buffers (see Table). Most notably, the otherwise weak
Affinity purification procedures involving interaction of an antibody
binding of mouse IgG1 is greatly improved.
with its antigen generally use binding buffers at physiologic pH
and ionic strength. However, many antibody purification methods Thermo Scientific Pierce Protein G IgG Binding Buffer uses sodium
do not use the antibody-antigen interaction; rather, they involve acetate (pH 5.0) to obtain the highest possible binding capacity of
binding of antibodies by immobilized ligands that are not the IgG to immobilized Protein G. The binding buffer for Protein A/G
antigen. In such cases, optimal binding conditions are determined is similar to our Protein A IgG Binding Buffer. The optimal binding
by the unique properties of the antibody-ligand interaction, which with Protein L occurs at pH 7.5; NAb Protein L Kits use phosphate
may be different from physiologic pH and ionic strength. buffered saline (PBS) as the binding buffer.
Once the binding interaction occurs (i.e., the antibody is “captured” Generally, a Pierce Binding Buffer is used by combining it 1:1 (v/v)
by the immobilized ligand), the support is washed with additional with clarified serum or ascites fluid. To avoid dilution, a sample
buffer to remove nonbound components of the sample. Finally, can be dialyzed into the recommended buffer. Purity of the sam-
elution buffer is added to break the binding interaction and release ples affects the total binding capacity of Protein A, G and A/G; total
the target molecule, which is then collected in its purified form. immunoglobulin binding capacities are higher for purified and con-
Elution buffer can dissociate binding partners by extremes of pH centrated antibodies than for crude serum or dilute samples.
(low or high), high salt (ionic strength), the use of detergents or
chaotropic agents that denature one or both of the molecules, Elution of antibodies that are bound to alphabet proteins, regardless
removal of a binding factor, or competition with a counter ligand. of the binding buffer used, is most generally accomplished using
In most cases, subsequent dialysis or desalting is required to 0.1 M glycine•HCl (pH 2–3) or other low pH buffer. In the vast majority
exchange the purified protein from elution buffer into a more of cases, this condition breaks affinity interactions without damaging
suitable buffer for storage or use. either the immobilized protein (allowing the affinity column to be
re-used) or the antibody. Our IgG Elution Buffer uses this acidic
Thermo Scientific Pierce IgG Binding and Elution Buffers have been (pH 2.8) condition. With this buffer, elution of IgG is usually sharp
optimized to provide the highest possible efficiency of IgG binding and complete. For example, nearly all bound IgG will elute in 3 ml
and elution using immobilized Protein A, Protein G and Protein A/G. of buffer from a 1 ml column of Protein A.
Use of other buffer formulations may significantly alter not only the
binding capacity but also the volumes of wash buffer required to Although brief exposure of antibody to acidic elution buffer usually
ensure good purification. is not harmful, it is advisable to neutralize the eluate as soon as
possible after its recovery to minimize the possibility of degradation.
General Binding and Elution Buffers for Protein A, G, A/G and L Our IgG Elution Buffer can be neutralized easily by adding 1/10th
Although Protein A, G and A/G bind immunoglobulins adequately volume of 1 M Tris•HCl, pH 7.5–9.0. Although long-term storage of
at physiologic pH and ionic strength (as with phosphate buffered the purified antibody in the neutralized buffer is possible, it is
saline, pH 7.2), optimal binding conditions are different for each common practice to dialyze or desalt into a buffer that is known
protein. For this reason, separate IgG Binding Buffers are available for to be suitable for storage.
use with each immobilized “alphabet protein” product. All our buffers
have long shelf lives and are premixed for maximum ease of use.
Binding capacities with different buffers expressed as mg of IgG bound per 2 ml of resin.
Immobilized Protein A Immobilized Protein G Immobilized Protein A/G

Serum Sample 0.1 M Tris•HCl Pierce Protein A 0.1 M Tris•HCl Pierce Protein G 0.1 M Tris•HCl Pierce Protein A
pH 8.0 Binding Buffer pH 8.0 Binding Buffer pH 8.0 Binding Buffer
Rabbit 17.81 33.19 21.51 27.75 13.89 19.61
Sheep 2.15 10.64 25.53 33.33 9.83 15.71
Bovine 6.16 22.76 31.72 48.10 15.13 22.06
Mouse 5.25 7.15 5.65 15.05 4.32 11.49
Rat 4.99 8.30 8.43 11.80 5.20 6.66
Horse 6.25 16.50 36.19 21.46 14.88 17.12
Dog 35.77 22.27 13.38 20.55 21.96 24.60
Chicken 0.91 1.21 1.63 7.27 1.21 4.10
Pig 29.61 24.83 21.25 27.51 19.24 29.48
Human 19.88 25.53 11.68 23.59 9.92 17.67

3 The Gentle Elution Buffer does not require neutralization and is
directly compatible with borate, citrate and acetate buffers,
IgG Bound (mg)
2 including our Protein G IgG Binding Buffer. However, the Gentle

Elution Buffer is not directly compatible with phosphate-containing
Protein A buffers, including our Protein A IgG Binding Buffer, with which it
1
Protein G will form an insoluble precipitate. For this reason, our Gentle
Protein A/G
Ag/Ab Binding Buffer, pH 8.0 is offered as a substitute for use with
0 Protein A.
4 5 6 7 8 9
Buffer pH Mouse IgG1 Mild Elution Buffer
A unique opportunity exists in Protein A with its weaker binding
Comparison of the binding characteristics of mouse IgG at various affinity to mouse IgG1 compared to other mouse IgG subclasses.
buffer pH levels. After binding total mouse IgG to immobilized Protein A using our
Protein A IgG Binding Buffer, Thermo Scientific Pierce Mouse IgG1
Gentle Ag/Ab Elution Buffer
Mild Elution Buffer can be used to selectively elute IgG1 without
Some antibodies are extremely labile and irreversibly denature
affecting the bound state of other IgG subclasses.
in the acidic conditions of the default Pierce IgG Elution Buffer.
Our Gentle Ag/Ab Elution Buffer is available for such situations. The buffer has a mild pH (6.0–6.1) to retain better biological
This near-neutral (pH 6.55) buffer dissociates affinity-bound activity in both the recovered antibody and the immobilized
immunoglobulins by ionic strength rather than by low pH. While Protein A. Neutralization or desalting of the collected IgG1 is not
being much less likely to degrade an antibody, it still retains necessary to retain activity. This advantage is especially important
excellent elution properties. when isolating potentially fragile monoclonal IgG1 antibodies. Because
the majority of mouse monoclonals are of the IgG1 subclass, this buffer
Our researchers have tested the effect of exposure to our Gentle
has many applications in the production of monoclonal antibodies.
Elution Buffer on monoclonal antibody activity. In one experiment,
three mouse monoclonals were incubated overnight in the Gentle After eluting the IgG1, other bound IgGs can be eluted using
Elution Buffer and then desalted. When analyzed in an ELISA standard IgG Elution Buffer. Our Protein A Binding Buffer and both
system, all three monoclonals retained full antigen-binding IgG and IgG1 Mild Elution Buffers are available as a kit. The system
capability as compared to untreated controls. enables quick, clean and mild isolation of mouse IgG1 from serum,
ascites or hybridoma culture supernatant.
Product # Description Highlights Pkg. Size

54200 Protein A/G IgG Binding Buffer • Ensures maximum recovery of IgG from immobilized Protein A/G 240 ml
21001 Protein A IgG Binding Buffer • High-yield isolation of Mouse IgG1 using Protein A columns 1L
21007 • Premixed and easy to use 3.75 L
21019 Protein G IgG Binding Buffer • Ensures maximum recovery of IgG from immobilized Protein G 1L
21011 3.75 L
21004 IgG Elution Buffer • High-yield isolation of IgG from Immobilized Protein A and Protein G 1L
21009 3.75 L
21020 Gentle Ag/Ab Binding Buffer pH 8.0 • Specially formulated and prefiltered 1L
21012 • Eliminates use of harsh acidic elution conditions 3.75 L
21030 Gentle Ag/Ab Elution Buffer pH 6.6 • Specially formulated for neutral elutions 100 ml
21027 • Not compatible with phosphate buffers 500 ml
21013 3.75 L
21016 IgM Binding Buffer • Specially formulated for optimal binding of mouse IgM 800 ml
21017 IgM Elution Buffer • Specially formulated for optimal recovery of mouse IgM 500 ml
21018 MBP Column Preparation Buffer • Specially formulated for use with Immobilized MBP and IgM Purification Kit 50 ml
21034 Mouse IgG1 Mild Elution Buffer • Separate IgG1 from other IgG subclasses 500 ml
21033 Mouse IgG1 Mild Binding and Elution Buffer Kit • Complete kit to allow mouse IgG1 to be separated from other mouse Kit
Includes: Pierce Protein A IgG Binding Buffer IgG subclasses 1L
Mouse IgG1 Mild Elution Buffer 500 ml
Pierce IgG Elution Buffer 1L
Melon Gel Purification Products Human Rabbit Goat

S M A1 A2 S M A1 A2 S M A1 A2
Thermo Scientific Melon Gel Products provide an exciting new
approach to purifying monoclonal and polyclonal antibodies from
serum, tissue culture supernatant and ascites fluid. Melon Gel
works with antibodies from a variety of species and subclasses,
many of which do not purify efficiently with Protein A or Protein G. H-
Because Melon Gel is not a bind-and-release support, it is
extremely fast and gentle to your antibodies, resulting in antibody
preparations of high purity and high activity! L-
How does it work?

Melon Gel contains a proprietary ligand that retains most protein
found in serum, ascites and culture supernatants, while allowing Thermo Scientific Melon Gel efficiently purifies IgG from human, rabbit, and
IgG to pass through the support and be collected in the flow- goat serum. Starting serum samples and resulting purification products were
through fraction. The resulting recovery and purity of the IgG electrophoresed by SDS-PAGE and stained with Thermo Scientific GelCode
isolated by this method rivals that obtained from the same samples Blue Stain. S = serum sample, M = Melon Gel-purified product, A1 and A2 =
successive elution fractions from Protein A purification. H and L denote antibody
using bind-and-release supports such as Protein A or Protein G. heavy and light chain bands in the reducing gel. Similar results were obtained
when comparing against Protein G purification.
Highlights:
• Simple, one-step protocol – no tedious binding, washing, and
90
multiple elution steps
80
• Rapid purification – purifies antibodies from serum four to six
times faster than Protein A or G methods 70
• High recovery and purity – antibodies from many species are 60

Percentage
recovered with greater than 90% yield and greater than 80% purity 50
• Robust purification – works with a wide range of antibodies 40
including many that do not purify well on Protein A or Protein G 30
• Gentle purification – No harsh elution conditions means 20
antibodies retain more activity
10
• Reusable support – Melon Gel Support can be used for multiple
0
antibody purifications Goat Human Rabbit
• Available in various formats – spin columns, purification kits and Species
chromatography cartridges for antibody purification from serum, Melon Gel Protein A Protein G
ascites and culture supernatant
Thermo Scientifc Melon Gel provides better purity than Protein A and G.
Several kits and package sizes of Melon Gel Resin are available. Percent purity was determined by purifying IgG from goat, human and rabbit
serum using Melon Gel, Protein A and Protein G. The products were separated
All sizes use the same formulation of Melon Gel Resin, Purification
by SDS-PAGE. Purity was calculated by determining the intensities of all bands
Buffer and Regenerant Solution but have slightly different protocols via fluorescence scanning and densitometry.
based on the most common application for each package size.
Components of any package size can be used at an appropriate
100
scale with any one of the procedures. Also available is a Tech
Tip for using Melon Gel Resin to remove BSA or gelatin from 90
commercially-supplied antibodies so that the antibody can be bio- 80
tinylated or otherwise labeled using amine-reactive chemistries. 70
60
Percentage
50
40
30
20
10
0
Human Mouse Rabbit
Species
Melon Gel Protein A Protein G
Thermo Scientific Melon Gel provides better recovery than Protein A and G.
Percent recovery was determined by applying human, mouse and rabbit IgG
to Melon Gel, Protein A and Protein G, and then comparing the absorbance at
280 nm of the original samples against that of the purified sample.

IgG purification performance of the Thermo Scientific Melon Gel System,
Protein A and Protein G. 1 2 3 4 5 6 7 8
Source Melon Gel Protein A Protein G

Human H H H
Transferrin
Mouse H H H
Albumin
Rabbit H H H
Heavy Chain
Rat H L M
Goat H L M
Cow M L H
Light Chain
Sheep M L H
Horse H L H
Guinea Pig H H L
Pig H H L
Thermo Scientific Melon Gel offers better recovery and purity from
Chicken N N N ascites fluid than Protein G. IgG1 was purified from ascites fluid, resolved by
Hamster H M M SDS-PAGE and detected with Thermo Scientific GelCode Blue Stain Reagent
(Product # 24590). Lane 1. Original ascites fluid, Lane 2. treated with Ascites
Donkey H M H Conditioning Reagent (Product # 45219) and purified with Thermo Scientific
H = high recovery, M = medium recovery, L = low recover, N = no recovery Melon Gel, Lane 3. Protein G column flow-through, Lanes 4–5. Protein G column
washes and Lane 6–8. Protein G column elutions. NOTE: The Protein G-purified
sample is contaminated with transferrin (Lanes 6–8). The Melon Gel-purified
1 2 3 4 5 6
sample is not (Lane 2).
Transferrin
Albumin
45206 Melon Gel IgG Spin Purification Kit† Kit
Heavy Chain Sufficient to purify up to 25 mg of IgG from serum.
Includes: Melon Gel 3 ml
Melon Gel Purification Buffer 100 ml
Spin Columns 27
Collection Tubes
45212 Melon Gel IgG Spin Purification Kit Kit

Light Chain Sufficient to purify up to 2 g of IgG from serum.
Includes: Melon Gel Support 5 ml
Melon Gel Purification Buffer 100X, dry mix
Melon Gel Regenerant
(dry mix; reconstitute to 1 L)
45214 Melon Gel Monoclonal IgG Purification Kit Kit

Thermo Scientific Melon Gel offers better recovery and purity from cell Sufficient to purify IgG from up to 1 L of cell culture
culture supernatant than Protein G. IgG was purified from cell culture supernatant or up to 200 ml of ascites fluid.
Includes: Melon Gel Support 200 ml
supernatant containing 10% fetal bovine serum (FBS), resolved by SDS-PAGE
Melon Gel Purification Buffer 100X, dry mix
and detected with Imperial Protein Stain† (Product # 24615). Lane 1. Original cell Melon Gel Regenerant 5X, dry mix
culture supernatant, Lane 2. IgG after precipitation with Saturated Ammonium
Sulfate (Product # 45216), Lane 3. Melon Gel-purified IgG and Lane 4–6. Protein 45216 Saturated Ammonium Sulfate Solution 1L
G-purified IgG. For use with the Melon Gel Monoclonal IgG
Purification Kit and Melon Gel IgG Purification Kits
for Sera or as a general-purpose salting-out agent
for protein-preparation applications.
45219 Ascites Conditioning Reagent 5 ml

For use with the Melon Gel Monoclonal IgG
Purification Kit.
Use of this reagent is described in the instructions
provided with Product # 45214.
89932 Pierce Chromatography Cartridges, Melon Gel 2 x 1

89933 Pierce Chromatography Cartridges, Melon Gel 1 x 5
45208 Melon Gel Spin Kit Plate 2 plates
96-well filter plate for IgG screening
89972 Melon Gel Purification Buffer 1 pack

89973 Melon Gel Regenerant 1 pack
† See patent information on inside back cover.
Thiophilic Gel Antibody Purification After use, our Thiophilic Adsorbent can be regenerated by treat-
ment with guanidine•HCl. Our data indicate that the Adsorbent
Thiophilic adsorption is a low-cost, efficient alternative to column can be used at least 10 times without significant loss of
ammonium sulfate precipitation for immunoglobulin purification binding capacity.
from crude samples. Ammonium sulfate precipitation must be
followed by several additional steps to completely remove Our Thiophilic Purification Kit includes 4 x 3 ml prepacked
contaminants in crude samples. Thiophilic adsorption is a simple, columns of Pierce Thiophilic Adsorbent, binding and elution
rapid, one-step method for antibody purification from serum, asci- buffers, column storage buffer, and guanidine•HCl for use in
tes or tissue culture supernatant. column regeneration. This simple, one-step method eliminates
the need for post-treatment of the sample before storage or
Thiophilic adsorption is a highly selective type of lyotropic subsequent conjugation to enzymes for use in immunoassays.
salt-promoted protein:ligand interaction phenomenon that has
been studied extensively by Porath and co-workers and other Suggested applications:
researchers.1 This interaction is termed thiophilic because it is • Efficient and selective isolation of immunoglobulins from human
distinguished by proteins that recognize a sulfone group in close serum under mild conditions1
proximity to a thioether. Thiophilic adsorption incorporates properties • Convenient and fast method for purification of mouse
of both hydrophobic and hydrophilic adsorption. However, in monoclonals from the culture media of cloned cells or from
contrast to strictly hydrophobic systems, thiophilic adsorption is ascites fluid2
not strongly promoted by high concentrations of sodium chloride. • Selective removal of immunoglobulins from fetal calf serum –
Instead, thiophilic adsorption is promoted by increased useful for cell culture in monoclonal antibody production3
concentrations of water-interacting, non-chaotropic salts such as • Rapid, straightforward procedure yielding essentially pure
potassium and ammonium sulfate. immunoglobulins from crude rabbit serum4
Thermo Scientific Pierce Thiophilic Adsorbent is 6% beaded • Purification of IgY from chicken5
agarose modified to contain simple sulfone/ thioether groups (see • Large-scale purification for biotechnology applications
structure at right). Our Thiophilic Adsorbent has a high binding
capacity (20 mg of immunoglobulin per ml of resin) and broad
Agarose
specificity toward immunoglobulins derived from various animal O S

Bead
species. Notably, thiophilic adsorption is one of few methods S OH

available for purification of IgY from chicken (see also subsequent O O
discussion of IgY purification). Among human serum proteins,
immunoglobulins and α2-macroglobulins are preferentially bound Structure of Thermo Scientific Pierce Thiophilic Adsorbent.
by our Thiophilic Adsorbent.2
References
Purification using Pierce Thiophilic Adsorbent results in good 1. Porath, J., et al. (1985). FEBS Lett. 185, 306–310.
2. Belew, M., et al. (1987). J. Immunol. Method 102, 173–182.
protein recovery with excellent preservation of antibody activity. 3. Hutchens, T.W. and Porath, J. (1987). Biochemistry 26, 7199–7204.
Sample preparation requires the addition of 0.5 M potassium 4. Lihme, A. and Heegaard, P.M.H. (1990). Anal. Biochem. 192, 64–69.
sulfate to the serum, ascites or culture fluid. Greater specificity 5. Unpublished internal Pierce documents.
for immunoglobulins is obtained if the sample is buffered at pH 8.0.
The gentle elution conditions (e.g., 50 mM sodium phosphate,
pH 7–8) yield concentrated, essentially salt-free, highly purified
immunoglobulins at near neutral pH.

References
Pierce Thiophilic Adsorbent and Purification Kit 1. Schulze, R.A., et al. (1994). Anal. Biochem. 220, 212–214.
Palmer, D.A., et al. (1994). Anal. Biochem. 222, 281–283.
Economical purification of mouse antibodies from ascites fluid. Porath, J., et al. (1985). FEBS Lett. 185, 306–310.
Hutchens, T.W. and Porath, J. (1986). Anal. Biochem. 159, 217–226.
Highlights: Belew, M., et al. (1987). J. Immunol. Method 102, 173–182.
Hutchens, T.W. and Porath, J. (1987). Biochemistry 26, 7199–7204.
• Binds to Fab and F(ab´)2 fragments Nopper, B., et al. (1989). Anal. Biochem. 180, 66–71.
• Binds to ScFv1 Harsay, E. and Schekman, R. (2002). J. Cell Biol. 156(2), 271–85.
Koustova, E. et al. (2001). J. Clin. Invest. 107(6), 737–44.
• High-capacity (20 mg/ml), good protein recovery and retention Suh, J.S., et al. (1998). Blood. 91(3), 916–22.
of antibody function
• Broad specificity toward immunoglobulins derived from various Ordering Information
animal species (see Table)
• Binds chicken IgY (also called IgG) Product # Description Pkg. Size
• Simple, rapid, one-step purification for monoclonal antibodies 20500 Pierce Thiophilic Adsorbent 10 ml
from ascites; easy to scale up 44916 †
Pierce Thiophilic Purification Kit Kit
• Used to enrich the immunoglobulin fraction from serum or tissue Includes: Thiophilic Adsorbent Columns 4 x 3 ml
culture supernatant Binding Buffer 1,000 ml
Elution Buffer 1,000 ml
• Efficient alternative to ammonium sulfate precipitation for Column Storage Buffer (2X) 100 ml
enriching antibodies from crude samples Guanidine•HCl Crystals 230 g
Column Extenders
• Gentle elution conditions yield concentrated, salt-free
immunoglobulin at near neutral pH † See patent information on inside back cover.
• High degree of purity
Binding characteristics of Thermo Scientific Pierce Thiophilic Adsorbent.
Total A280 Bound

Species from 1 ml Serum % Purity by HPLC
Human 4.8 70
Mouse 8.6 63
Mouse IgG1 11.6 92
Mouse IgG2a 9.3 88
Mouse IgG2b 9.8 97
Mouse IgG3 10.7 94
Rat 13.0 79
Bovine 17.9 90
Calf 11.1 89
Chicken 5.2 76
Dog 12.2 91
Goat 17.3 92
Guinea Pig 11.1 71
Horse 13.0 93
Pig 21.1 90
Rabbit 6.7 84
Sheep 12.3 89
IgM Purification IgM purification with Pierce Immobilized Mannan Binding Protein
is temperature- and calcium-dependent. Binding and washing
Structure of IgM steps are performed at 4°C in 10 mM Tris•HCl (pH 7.4) buffer con-
IgM is a high molecular mass glycoprotein (900–950 kDa) with a taining sodium chloride and 20 mM calcium chloride. Elution is
carbohydrate content of approximately 12%. This antibody is found made at room temperature in a similar Tris buffer, except that it
at concentrations of 0.5–2 mg/ml in serum.1 In vivo, IgM has a half contains EDTA and is devoid of calcium chloride. An Immobilized
life of five days, and its catabolism is two- to three-fold greater MBP Column can be regenerated at least 10 times with no appar-
than that of IgG. ent loss of binding capacity.
In the sera of mammals, birds and reptiles, IgM has a pentameric Immobilized MBP is available in both beaded agarose and
structure. However, mouse and human IgM structures differ in the UltraLink Biosupport formats. Binding, elution and column prepara-
location of disulfide bridges that link monomers together to form tion buffers are also available. The IgM Purification Kit contains
the pentamer (Figure).2 Disulfides are arranged in series in mouse sufficient buffers to perform 10 purifications using a 5 ml column of
IgM and in parallel in human IgM. Immobilized MBP. The kit is easy to use and yields 90% pure
mouse IgM (from ascites) with a very simple protocol.
Challenges to IgM Purification
Protein A binds IgM poorly, in part because binding sites on the Fc
region of the monomers are sterically hindered by the pentameric
structure of IgM. Until recently, no readily available affinity
chromatography product existed for one-step IgM purification.
Standard methods for IgM purification generally are multi-step,
tedious processes or they are not effective for removing all of the
major impurities present in IgM samples.3
Traditionally, IgM was purified by ammonium sulfate precipitation

followed by gel filtration, ion exchange chromatography or zone
electrophoresis.4 Other methods that have been used include
use of DEAE cellulose,5 immobilized DNA6 and a combination of
ammonium sulfate precipitation and subsequent removal of IgG Human IgM
with Protein A or G.3
Nethery, et al. developed an IgM affinity purification method

using C1q, a 439 kDa complement component that recognizes
carbohydrate on cell surfaces.7 This temperature-dependent
binding method yielded relatively pure IgM. However, co-purification
of IgG was a problem, and C1q is expensive and difficult
to purify.
Immobilized Mannan Binding Protein

To develop an effective affinity matrix, our scientists examined
C1q and another similarly structured protein, mannan binding
protein (MBP). Serum MBP, like C1q, is capable of initiating
carbohydrate-mediated complement activation. MBP is a mannose
and N-acetylglucosamine-specific lectin found in mammalian sera, Mouse IgM
and it has considerable structural homology to C1q.8 MBP subunits Structure of IgM, adapted from Matthew and Reichardt.8
are identical, each with molecular mass of approximately 31 kDa
(C1q has six each of three different polypeptide subunits of molec- References
1. Milstein, C., et al. (1975). Biochem. J. 151, 615–624.
ular mass 24–28 kDa). Studies in our labs show that MBP does not 2. Coppola, G., et al. (1989). J. Chromatogr. 476, 269–290.
bind F(ab´)2 and Fab. 3. Fahey, J. and Terry, E. (1967). Handbook of Experimental Immunology,
Chapter 8. D.M. Weir, Ed. Blackwell, Oxford, U.K.
We have developed an easy-to-use Thermo Scientific Pierce 4. Cambier, J. and Butler F. (1974). Prep. Biochem. 4(1), 31–46.
5. Abdullah, M., et al. (1985). J. Chromatogr. 347, 129–136.
Immobilized Mannan Binding Protein and Buffer System to purify 6. Nethery, A., et al. (1990). J. Immunol. Method 126, 57–60.
IgM. It is most effective for purifying mouse IgM from ascites. 7. Ohta, M., et al. (1990). J. Biol. Chem. 264, 1980–1984.
8. Matthew, W. and Reichardt, L. (1982). J. Immunol. Method 50, 239–253.
Purified IgM can be obtained from a single pass over the affinity
column. Human IgM will bind to the support, albeit with slightly
lower capacity, and yield a product at least 88% pure as assessed
by HPLC. The purification of IgM from other species and mouse
serum has not yet been optimized.

Immobilized MBP and IgM Purification Kit IgA Purification
Easy IgM purification with guaranteed 88% pure mouse IgM! Human IgA Purification
Jacalin is an α-D-galactose binding lectin extracted from jack-fruit
100 seeds (Artocarpus integrifolia). The lectin is a glycoprotein of
IgM
approximately 40 kDa composed of four identical subunits. Jacalin
immobilized on supports such as agarose has been useful for
80
the purification of human serum or secretory IgA1. IgA can be
Absorbance at 280 nm
separated from human IgG and IgM in human serum or colostrum.1

(percent of max.)
60 IgD is reported to bind to jacalin.2 Immobilized jacalin is also useful

for removing contaminating IgA from IgG samples.
40 Binding of IgA to immobilized jacalin occurs at physiologic pH
and ionic strength, as in phosphate buffered saline (PBS). Elution
20 of bound IgA occurs with competitor ligand (e.g., 0.1 M melibiose
or 0.1 M α-D-galactose) in PBS. We offer immobilized jacalin on
crosslinked 6% agarose.
0
0 10 20 30 40 50 References
Time (min.) 1. Roque-Barreira, M.C. and Campos-Neto, A. (1985). J. Immunol. Method 134(30), 1740–1743.
2. Aucouturier, P., et al. (1987). Mol. Immunol. 24(5), 503–511.
Demonstration of the high purity of MBP-purified IgM from mouse ascites.

The bound material from mouse ascites was eluted from the 5 ml MBP column Immobilized Jacalin
as described in the Standard Protocol. The highest 280 nm absorbing fraction
from the elution was chromatographed using the conditions described in the
instructions.
Ideal for human IgA purification.
References Highlights:
Nethery, A., et al. (1990). J. Immunol. Method 126, 57–60. • Ideal for preparing human IgA that is free of contaminating IgG
Ohta, M., et al. (1990). J. Biol. Chem. 265, 1980–1984.
Nevens, J.R., et al. (1992). J. Chromatogr. 597, 247–256. • Found to bind human IgA1, but not human IgA2 – useful for
separating the two subclasses
Ordering Information References
Kumar, G.S., et al. (1982). J. Biosci. 4, 257–261.
Product # Description Pkg. Size Roque-Barreira, M.C. and Campos-Neto, A. (1985). J. Immunol. 134, 1740–1743.
Mestecky, J., et al. (1971). J. Immunol. 107, 605–607.
22212 Immobilized Mannan Binding Protein 10 ml Van Kamp, G.J. (1979). J. Immunol. Method 27, 301–305.
Capacity: ~1 mg IgM/ml of resin Kondoh, H., et al. (1986). J. Immunol. Method 88, 171–173.
44897 IgM Purification Kit Kit

Includes: Immobilized MBP Column 5 ml Ordering Information
IgM Binding Buffer 800 ml
IgM Elution Buffer 500 ml
MBP Column Preparation Buffer 50 ml Product # Description Pkg. Size
Column Extender
20395 Immobilized Jacalin 5 ml
21016 IgM Binding Buffer 800 ml Capacity: 1–3 mg human IgA/ml of resin
21017 IgM Elution Buffer 500 ml Loading: 4.5 mg of jacalin/ml of resin
21018 MBP Column Preparation Buffer 50 ml
53123 UltraLink Immobilized Mannan Binding Protein 5 ml
Capacity: > 0.75 mg IgM/ml of resin
Chicken IgY Purification Pierce Chicken IgY Purification Kit

Properties of IgY Purifies 100 mg of chicken IgY with higher purity than ever before!
Chickens produce a unique immunoglobulin molecule called IgY.
There are several advantages to production and use of IgY over Highlights:
mammalian immunoglobulins. With regard to production, raising • More for your money – purifies twice the amount of IgY as the
and immunizing chickens is relatively simple, chickens are more leading competitor’s kit with a lower cost-per-mg of IgY purified
likely to produce an immune response to conserved mammalian • Higher purity – 85–95% by SDS-PAGE analysis (see Figure)
protein antigens, and chickens produce 15- to 20-times more • Ease-of-use – the simple precipitation method works without
antibody than rabbits. affinity columns
• Flexibility – eggs can be stored in buffer and purified at a later date
Most importantly, IgY is naturally packaged at high concentrations
in egg yolks, making repeated collection of antibody from • Convenience – use eggs directly out of the refrigerator; no need
immunized hens noninvasive. A single egg yolk from an immunized to wait for them to warm up
chicken contains approximately 300 mg of IgY. Whole eggs or
separated egg yolks can be collected and stored frozen for later
extraction of antibody.
Other advantages of IgY for use in immunoassays are that it does Product # Description Pkg. Size
not bind rheumatoid factor or other anti-mammalian IgGs, does not 44918 Chicken IgY Purification Kit Kit
activate complement, and generally has much lower probability of Sufficient reagents to purify 5 egg yolks.
Includes: Pierce Delipidation Reagent 500 ml
nonspecific binding to mammalian tissues and extracts. Pierce IgY Precipitation Reagent 500 ml
Pierce Egg Separator 1
IgY Purification Methods
44922 Chicken IgY Purification Kit Kit
One challenge with regard to IgY is that it can be difficult to purify. Sufficient reagents to purify 25 egg yolks.
Protein A, Protein G and other Fc-binding proteins do not bind IgY.
21055 Delipidation Reagent 500 ml
Thermo Scientific Pierce Thiophilic Adsorbent (see page 48) 21057 IgY Precipitation Reagent 500 ml
enables moderate yields of fairly pure IgY from serum and other 21060 Egg Separator 1
fluids. However, complete procedures for our Thiophilic Adsorbent
have not been developed for use with egg yolks, which have very high
lipid concentrations. SDS-PAGE analysis of Thermo Scientific Pierce Chicken IgY purification.
MW Markers
Thermo Scientific Pierce Chicken IgY Purification Kits were spe-

IgY Control
Supplier P
Pierce Kit
cifically developed for efficient purification of IgY from egg yolks.

After separating an intact yolk from egg white using an egg sepa-
rator, Thermo Scientific Pierce Delipidation Reagent is added to kDa
separate the proteins from lipid. The delipidation reagent can also 150
be used to store an egg yolk in the freezer for up to one year. After
delipidation, the protein-containing sample fraction is mixed with
100
Thermo Scientific Pierce IgY Precipitation Reagent to create a
relatively pure IgY precipitate that is recovered by centrifugation.
75
Routinely, 80-120 mg of high purity (> 85%), intact IgY can be
50
obtained per egg using the Pierce Kit.
References
1. Cassidy, P.B., et al. (2006). Carcinogenesis. 27, 2538–2549. 35
2. Kamiya, Y., et al. (2005).J. Biol. Chem. 280, 37178–37182.
3. Kantardzhieva, A. et al. (2005). Retinal Cell Biol. 46, 2192–2201. 25
15
Chicken IgY was purified according to each manufacturer’s instructions.

The gel shows the analysis of 2 µg of protein applied per well. The Pierce IgY
Kit purified the chicken IgY to a purity level of > 85% using Thermo Scientific
GelCode Blue Stain Reagent (Product # 24590). The competitor’s product
achieved only a 53% purity level. The arrow indicates intact IgY.

Affinity Purification of Specific Antibodies Little variation exists among typical binding and elution conditions
for affinity purification of antibodies because at the core of each
Although Proteins A, G, A/G and L are excellent ligands for procedure is the affinity of an antibody for its respective antigen.
purification of total IgG from a sample, purification of an antibody Since antibodies are designed to recognize and bind antigens
specific for a particular antigen and free of contamination from tightly under physiologic conditions, most affinity purification
other immunoglobulins is often required. This can be accomplished procedures use binding conditions that mimic physiologic pH and
by immobilizing the particular antigen used for immunization so ionic strength. The most common binding buffers are phosphate
that only those antibodies that bind specifically to the antigen are buffered saline (PBS) and Tris buffered saline (TBS) at pH 7.2 and
purified in the procedure. Activated affinity supports that can be 1.5 M NaCl (see pages 4–5 for convenient premixed buffer packs).
used to immobilize peptides or other antigens for use in affinity Once the antibody has been bound to an immobilized antigen,
purification are described later on pages 10–25. additional binding buffer is used to wash unbound material from
the support. To minimize nonspecific binding, many researchers
Successful affinity purification of antibody depends on effective use wash buffer containing additional salt or detergent to disrupt
presentation of the relevant epitopes on the antigen to binding any weak interactions.
sites of the antibody. If the antigen is small and immobilized
directly to a solid support surface by multiple chemical bonds, Specific, purified antibodies are eluted from an affinity resin by
important epitopes may be blocked or sterically hindered, prohibiting altering the pH or ionic strength of the buffer (see page 3 for
effective antibody binding. Therefore, it is best to immobilize recipes of common elution buffers). Antibodies generally are
antigens using a unique functional group (e.g., sulfhydryl on a resilient proteins that tolerate a range of pH from 2.5 to 11.5 with
single terminal cysteine in a peptide) and to use an activated minimal loss of activity, and pH-shift is by far the most common
support whose reactive groups occur on spacer arms that are elution strategy. In some cases an antibody-antigen interaction is
several atoms long. For larger antigens, especially those with not efficiently disrupted by pH changes or is damaged by the pH,
multiple sites of immobilization, the spacer arm length becomes requiring that an alternate strategy be employed.
less important since the antigen itself serves as an effective
spacer between the support matrix and the epitope. Generally, See section on Covalent Coupling of Affinity Ligands to Solid Supports (page 10) for
more information on immobilization of antigens.
if the antigen was crosslinked to a carrier protein to facilitate
antibody production, best results are obtained when the antigen
is immobilized for affinity purification using the same chemistry
(e.g., reaction to primary amines, sulfhydryls, carboxylic acids or
aldehydes). In this way, all epitopes will be available for antibody
binding, allowing greater efficiency in purification and recovery of
the specific immunoglobulin.
Immunoprecipitation and Co-Immunoprecipitation Assays
Co-Immunoprecipitation
Immunoprecipitation can be extended to yet another level of
affinity purification. In co-immunoprecipitation (co-IP), an antibody
is used to capture not only a specific antigen, but also whatever
protein or complex is bound to the antigen. When Protein A or
Protein G is used to affinity-purify the antibody:antigen complex,
there is a minimum of three levels of affinity binding interaction
involved in co-IP. For a co-IP to be successful, the binding buffer
conditions must be compatible with all levels of binding interaction
involved, and the epitope on the antigen must not be blocked by
protein:protein interactions.
The complexity of a co-IP system can be reduced by immobilizing

the antibody directly and covalently to a resin. This simplifies the
binding conditions but, more importantly, it allows the precipitated
complex to be eluted from the resin while the antibody remains
intact and immobilized. When the precipitated complex is
analyzed by SDS-PAGE or Western blotting, it is free from
Immunoprecipitation interfering antibody heavy- and light-chain and the analysis is
greatly simplified.
Immunoprecipitation (IP) refers to the small-scale affinity
purification of antigen using a specific antibody. Classical
immunoprecipitation involves the following steps:
1. Incubate specific antibody with an antigen-containing sample.

2. Capture antibody-antigen complex with Protein A or Protein G
agarose resin (Protein A or Protein G binds the antibody, which
is bound to its antigen).
3. Wash the resin with buffer to remove non-bound sample
components.
4. Elute the antigen (and antibody) by boiling the resin in reducing
SDS-PAGE sample buffer.
5. Analyze eluted sample by gel electrophoresis.
Classical IP is usually performed in a microcentrifuge tube with

0.1–1 ml of an antigen-containing sample using 10–50 µl of Protein A
or Protein G agarose. The beaded resin is pelleted by centrifuga-
tion after each step (washes and elution) and the supernatant is
removed. It is practically impossible to identify an elution buffer
that will elute antigen from the antibody without also eluting the
antibody from Protein A or Protein G. Therefore, the eluted sample
will always contain both antigen and antibody, and reducing gel
electrophoresis of the eluted sample will yield both the antigen
band and heavy- and light-chain antibody fragment bands.
To avoid antibody contamination of the eluted antigen, modifications

to the classical IP can be made so that the antibody is permanently
immobilized and will not elute with the antigen. One strategy is to first
bind the antibody to the Protein A or Protein G resin and then cova-
lently crosslink the antibody to the Protein A or Protein G. Seize X
Immunoprecipitation Kits use this approach. Another strategy is to
directly couple antibody to an activated affinity support such as
AminoLink Plus Coupling Resin (see pages 11 and 14). Seize Primary
Immunoprecipitation Kits use this approach. Also, because Thermo
Scientific Pierce IP Kits use our Spin Cup Columns, they improve
reproducibility of IP experiments by eliminating resin loss during the
washing procedures.

Thermo Scientific Products for Immunoprecipitation
and Co-Immunoprecipitation Assays
Pierce Classic Immunoprecipitation Kits
Agarose
Bead
Protein
For fast, easy recovery of immune complexed proteins. G
Highlights:
• Improve assay consistency – our Spin Cups eliminate resin loss
• Easy protocol – immunoprecipitate (IP) out the target protein Capture Antibody-Antigen Complexes
from crude lysate in just four easy steps with Protein A or G Agarose Beads
• Fast – IP a protein using Pierce Spin Cups and tubes in less than
one hour How it works
Our Classic Immunoprecipitation Kits use our Spin Cups and
• Economical – reuse the Immobilized Protein A or Protein G
Microcentrifuge Collection Tubes for easy separation of the solid
support for future IPs
Protein A or Protein G support from the recovered protein. There
• Complete kits – choose kits with or without cell lysis reagents is no need to carefully pipette supernatant away from the support.
for bacterial, mammalian or yeast cells Add the sample containing the immune complex to the Protein A or
G support and centrifuge to remove nonbound materials. Recover
Step 1. Incubate antibody Step 2. Apply sample to the immunoprecipitated protein by adding elution buffer and then
with cell lysate Protein A or G Agarose Resin spinning. Next, mix the eluted protein with the sample loading
buffer supplied in the kit and analyze by SDS-PAGE.
Thermo Scientific Pierce Spin Cup
and Microcentrifuge Tube
Form Immune Complex
45213 Classic Protein A IP Kit Kit
Step 3. Centrifuge to remove nonbound proteins Sufficient reagent for 50 immunoprecipitations.
Includes: Protein A Plus Agarose 1 ml
Sample Loading Buffer 5 ml
Binding Buffer 500 ml
Elution Buffer 50 ml
Pierce Spin Cups 12/pkg.
Pierce Microcentrifuge Tubes 72/pkg.
Centrifuge
1 minute 45218 Classic Protein G IP Kit Kit
Sufficient reagent for 50 immunoprecipitations.
Includes: Protein G Plus Agarose 1 ml
Add Wash Buffer Nonbound Proteins Binding Buffer 500 ml
Step 4. Elute bound proteins (immune complex) Pierce Microcentrifuge Tubes 72/pkg.
45217 Classic Mammalian IP Kit Kit

Sufficient reagent for 50 immunoprecipitations.
Includes: Protein G Plus Agarose
Binding Buffer 5 ml
Centrifuge
1 minute Pierce Spin Cups 250 ml
M-PER Mammalian Protein 72/pkg.
Extraction Reagent 25 ml
Add Elution Buffer Immunoprecipitated Protein
69702 Spin Cups – Cellulose Acetate Filter 50 units
69720 Microcentrifuge Tubes, 2 ml 72 tubes
Step 5. Analyze proteins on Western blot
MW Lysate
Marker
The Thermo Scientific Pierce Classic Immunoprecipitation Kit Protocol.
Seize X Immunoprecipitation Kits

DSS
Recover more protein without antibody interference!
Agarose
Agarose
Crosslinker
Bead
Bead
Protein Protein
G G
Seize X Kits extend the functionality of traditional immunoprecipitation
(IP) methods by adding crosslinking technology and microcentrifuge
spin cup sample handling to the procedure. The primary benefits
resulting from these features are the ability to purify target protein Immobilized Protein G Disuccinimidyl Suberate Oriented and Covalently
Binds Antibody Crosslinks Proteins Immobilized Antibody
without contamination by the antibody and the ability to more effec-
tively wash and separate samples from the beaded agarose resin.
Highlights: How it works

• No antibody contamination – no elution of antibody heavy and light The Seize X IP method involves capturing the IP antibody to
chains to interfere in SDS-PAGE analysis of purified protein Protein A or Protein G beaded agarose resin and covalently
• Improved assay reliability and sample handling – our Spin Cups immobilizing it to the support by crosslinking with our
eliminate resin loss and provide for more efficient separation of disuccinmidyl suberate (DSS) reagent (see Figure). The antibody
solutions compared to traditional IP resin is then incubated with the sample that contains the protein
• Potentially more economical – the prepared antibody affinity resin antigen of interest, allowing the antibody:antigen complex to form.
can be reused multiple times to immunoprecipitate a greater total After washing to remove nonbound sample components, the
amount of protein than in traditional IP antigen is recovered using the elution buffer supplied in the kit.
• Complete kits – package includes sufficient reagents and spin The entire procedure is performed in a microcentrifuge spin cup,
cups to immobilize at least four different antibody samples, each allowing solutions to be fully separated from the agarose resin
of which may be reused multiple times for a total of about forty (40) upon brief centrifugation. Only antigen is eluted by the procedure
IP experiments (see Figure), enabling it to be identified and further analyzed without
interference from antibody fragments. Furthermore, the antibody
1 2 3 resin can be reused for additional rounds of immunoprecipitation.
kDa
References
Ikemoto, A., et al. (2003). J. Biol. Chem. 278, 5929–5940.
Kerkela, R., et al. (2002). J. Biol. Chem. 277, 13752–13760.
215 Parkin, S.E., et al. (2002). J. Biol. Chem. 277, 23563–23572.
Quill, T.A., et al. (2001). Proc Nat. Acad. Sci. USA 98, 12527–12531.
Roti, E.C., et al. (2002). J. Biol. Chem. 277, 47779–47785.
120 Sklyarova, T., et al. (2002). J. Biol. Chem. 277, 39840–39849.
84
60
39 45215 Seize X Protein A Immunoprecipitation Kit Kit

Sufficient reagents to immobilize 4 different antibodies
28 and perform 40 total IP reactions.
Includes: Protein A Plus Agarose 1 ml
18.3 Binding/Wash Buffer 500 ml
DSS 8 x 2 mg
Thermo Scientific Seize X IP Kits purify antigen without interference by the IP
antibody. E. coli cells expressing fusion of green fluorescent protein (GFP) were 45210 Seize X Protein G Immunoprecipitation Kit Kit
extracted with B-PER Reagent† (Product # 78248) and then immunoprecipitated Sufficient reagents to immobilize 4 different antibodies
using a polyclonal goat anti-GFP antibody. Eluted IP products were separated and perform 40 total IP reactions.
by SDS-PAGE and coomassie stained (Product # 24590) to detect total protein. Includes: Protein G Plus Agarose 1 ml
Lane 1: single product obtained using the Seize X Protein G IP Kit. Lane 2: antigen Binding/Washer Buffer 500 ml
and antibody fragments resulting from traditional IP method. Lane 3: molecular Sample Loading Buffer 5 ml
weight marker. DSS 8 x 2 mg
45225 Seize X Mammalian Immunoprecipitation Kit Kit

Same scale and components as Product #45210
and also includes:
M-PER Mammalian Protein 25 ml
Extraction Reagent
69702 Pierce Spin Cups – Cellulose Acetate Filter 50 units

69720 Pierce Microcentrifuge Tubes, 2 ml 72 tubes
† See patent information on inside back cover.

Seize Primary Immunoprecipitation Kits Y
Agarose
O H2N
Bead
Y
+
Y
No species or subclass requirements and no antibody band interference. C
Y
H2N Bead
H NH2
NH2 Y Y
Y
Seize Primary Immunoprecipitation Kits eliminate the need to
determine if an antibody species or subclass binds well to Protein
Thermo Scientific
A or Protein G. Using this kit, directly couple a primary antibody AminoLink Plus Resin Antibody with Covalently
to the AminoLink Plus Activated Support to create a reusable, (Aldehyde Activated) Primary Amines Immobilized Antibody
immunoprecipitation (IP) resin that prevents the antibody from
co-eluting with the target protein. How it works
Both Seize Primary and Seize X IP Kits offer a reusable antibody-
Highlights: coupled resin resulting in no antibody heavy and light chain con-
• No antibody contamination – antibody heavy and light chains tamination. However, in the Seize X IP Procedure, DSS is used to
do not elute with purified protein; eliminates interference from crosslink the antibody to the Protein A or Protein G resin. Because
antibody heavy and light chains in SDS-PAGE or Western blots crosslinking often reduces the antibody-binding capacity, this step
• IP with any species and subclass of antibody – use chicken IgY, has been eliminated in the Primary IP Kit to allow for greater recovery
human IgE, mouse IgM or any purified, carrier-free protein of the target protein. While the Classic Kit (page 55) method potentially
• Prepared antibody affinity resin is reusable for multiple rounds yields a greater quantity of target protein, the presence of antibody
of IP – represents a possible savings of expensive antibody heavy and light chains in the eluent can distort or hide the recovered
• Convenient sample handling – our Spin Cups eliminate resin target protein band on a polyacrylamide gel.
loss and provide for efficient separation of solutions References
• Highest possible yield – retains antibody function better than Kwon, Y.H., et al. (2002). J. Biol. Chem. 277, 41417–41422.
Eberhardt, W., et al. (2002). Mol. Endocrinol. 16, 1752–1766.
other covalent attachment methods (e.g., Seize X Kits)
• Complete kits – package includes sufficient reagents and spin
cups to immobilize at least four different antibody samples, each Ordering Information
of which may be reused multiple times for multiple IP experiments
100 45335 Seize Primary Immunoprecipitation Kit Kit
Sufficient reagents to immobilize 10 different antibodies
and perform 20 total IP reactions.*
Percent Coupled
Includes: AminoLink Plus Coupling Resin 2 ml

80
Goat Coupling Buffer 500 ml
Quenching Buffer 60 ml
Human
Wash Buffer 60 ml
Rabbit 1 Reducing Agent 0.5 ml
60
Rabbit 2 Pierce Microcentrifuge Tubes 150 each
Mouse Pierce Spin Cups 12/pkg.
Chicken Binding Buffer 500 ml
40 Immunoprecipitation Sample Buffer 500 ml
0 1 2 3 4 5 Elution Buffer 50 ml
Time (Hours) Lane Marker 5 ml
Sample Buffer, Non-reducing
Antibody coupling efficiency of mammalian and avian antibody. Purified
45332 Seize Primary Mammalian Kit
antibody (200 µg) from various species was coupled to 200 µl of Thermo Immunoprecipitation Kit
Scientific AminoLink Plus Coupling Resin at 1-hour intervals for 4 hours at Same scale and components as Product #45335
room temperature. For the chicken antibody, 500 µg was used. and also includes:
Extraction Reagent
Flow-through
MW Marker
69702 Pierce Spin Cups – Cellulose Acetate Filter 50 units

Elution 1
Elution 2
Elution 3
Elution 4
Wash 1
Wash 2
Wash 3
Lysate
69720 Pierce Microcentrifuge Tubes, 2 ml 72 tubes

* Based on a scale of 100–200 µl of settled resin per IP reaction. Kit can provide up
to 200 IP reactions if more Pierce Spin Cups are purchased separately.
The Thermo Scientific Seize Primary Kit produces exceptionally clean IP results.
Seize Coated Plate Immunoprecipitation Kits Choosing between Protein G, Protein A/G and Streptavidin
Coated Plate Kits
Pre-coated 96-well plates are easier to use and faster than Streptavidin is a protein that binds specifically and very strongly
traditional microcentrifuge tube methods. to biotin; therefore, the Streptavidin Coated Plate IP Kit (#45360) is
appropriate for immunoprecipitation when using a biotin-labeled
Thermo Scientific Seize Coated Plate IP Kits provide for rapid (biotinylated) antibody. In fact, this kit can be used to affinity-purify
immunoprecipitation of multiple samples without the usual tedium a binding partner to any antibody species or subclass or any other
of pipetting, centrifuging and separating beaded affinity resin in protein or molecule that is biotinylated. Because the streptavidin-
individual microcentrifuge tubes. Immunoprecipitation is accom- biotin affinity interaction is so strong, the elution step generally will
plished using coated 96-well microplates rather than beaded dissociate only the antigen (binding partner), not the biotinylated
agarose resin. The plate format allows for faster processing of antibody or “bait” protein. See page 26 for more information about
multiple samples. Select from Protein A/G-, Protein G- or streptavi- avidin:biotin binding.
din-coated plates.
Protein A and Protein G are different proteins that bind to immuno-
Highlights: globulins (primarily only IgG). Typically, Protein A is preferred for
• Ready-to-use, high quality coated plates provide high capacity use with Rabbit polyclonal antibodies, while Protein G is preferred
and consistency for use with mouse antibodies (especially monoclonals of the IgG1
• Plate format best suited for simultaneously processing multiple subclass). Protein A/G is a recombinant of Protein A and Protein G
samples and their control conditions that has the additive binding properties of both proteins. See
• Faster, easier and more thorough washing than with traditional page 37 for more information about Protein A, G and A/G.
tube/resin IP methods Reference
• Uses familiar and convenient ELISA tools (multichannel pipettors Desai, S. and Hermanson, G. (1997). Previews 1(3), 2–7.
and plate washing); no tedious separation of supernatant from
pelleted resin beads, and no tubes to open and close and Ordering Information
centrifuge
• Coated plates are 96-well strip plates, convenient for experiments Product # Description Pkg. Size
requiring only a partial plate 45350 Seize Protein A/G Coated Plate IP Kit Kit
• Easy-to-follow instructions, including detailed explanation of Antibody binding capacity/well: 2.5 µg.
Sufficient capacity for downstream analysis
appropriate controls of IP or co-IP proteins in-gel or by Western blot.
• Three kits available, suitable for most common antibody types Includes: Protein A/G Coated 12 x 8-Well Strip Plates 2 plates
(mouse, rabbit, human and goat IgG subclasses) or any Phosphate Buffered Saline 2 packs
Surfact-Amps X-100 (10% Triton X-100) 6 x 10 ml
biotinylated antibody or “bait” protein Elution Buffer 50 ml
Uncoated 96-well Strip Plates (white), 2 each
1 2 3 4 5 (for sample collection and neutralization)
Thermo Scientific Seize Protein G Plate Sealers 18 sheets
Coated Plate IP Kit (Product # 45355) 45360 Seize Streptavidin Coated Plate IP Kit Kit
immunoprecipitation of CD71 (trans- Antibody binding capacity/well: 5 µg.
ferring receptor) from human serum Sufficient capacity for downstream analysis
using and a goat anti-CD71 poly- of IP or co-IP proteins in-gel or by Western blot.
clonal antibody. Eluted products for Includes: High Binding Capacity Streptavidin
the experimental and control samples Coated Plates 2 plates
were mixed with nonreducing sample Biotin Blocking Buffer 30 ml
loading buffer, separated by SDS- Phosphate Buffered Saline 2 packs
Surfact-Amps X-100 (10% Triton X-100) 6 x 10 ml
PAGE, transferred to nitrocellulose Elution Buffer 50 ml
membrane and detected by Western Neutralization Buffer 7 ml
blotting with the IP antibody, Goat- Uncoated 96-well Strip Plates (white), 2 each
anti-mouse-HRP conjugated second- (for sample collection and neutralization)
ary antibody (Product # 31432) and Plate Sealers 18 sheets
Thermo Scientific SuperSignal West
Dura Chemiluminescent Substrate
(Product # 34076).
Lane 1: Experiment (immunoprecipitation product)

Lane 2: Antibody-only control (no sample)
Lane 3: Human serum sample control (no antibody)
Lane 4: Plate control (no antibody or human sample)
Lane 5: Pure target protein (CD71) for size reference

Pierce Co-Immunoprecipitation Kits
Highlights:
All the components needed to perform a properly controlled • AminoLink Plus Coupling Resin for covalent attachment of
co-IP experiment. primary antibody prevents co-elution of antibody or antibody
fragments with the target protein interactors
Co-immunoprecipitation, or co-IP as it is widely known, is the
gateway method to all other in vitro methods for protein interaction • Couple any purified antibody regardless of species or Ig subclass
analysis. As a result, co-IP is among the most common techniques (chicken IgY, human IgE, mouse IgG1, IgM, etc.)
for protein:protein interaction discovery and verification. For • Physiologic co-IP buffer for optimal binding
example, protein interactions discovered through the use of yeast • Control gel included – allows for a properly controlled co-IP
two-hybrid experiments are most often confirmed by co-IP experiment
experiments. The Thermo Scientific Pierce Co-Immunoprecipitation • Spin cup columns for efficient washing and elution of small
Kits were configured to provide the essential tools to effectively gel volumes
perform a co-IP experiment using the primary antibody of your • Coupled antibody support reusable up to 10 times – saves
choice. These kits provide those attempting a co-IP for the first precious antibody.
time, as well as those experienced in the method, an ideal
system for designing, constructing and performing a controlled co-
IP experiment.
Benefits of target antibody immobilization Product # Description Pkg. Size

Our Co-Immunoprecipitation Kit utilizes Thermo Scientific 23600 Pierce Co-IP Kit Kit
AminoLink Plus Coupling Resin, which brings several benefits to Sufficient material to immobilize up to 10 antibodies
and perform a minimum of 40 co-IP reactions if
the co-IP application: 25 µl of antibody immobilized support is used.
More co-IPs can be performed if smaller volumes
• The AminoLink Coupling Resin allows a purified antibody to are used.
Includes: AminoLink Plus Coupling Resin 2 ml
be directly and covalently immobilized onto the matrix while (4 ml of a 50% slurry)
retaining antibody activity Control Resin, (4 ml of a 50% slurry) 2 ml
BupH Modified Dulbecco’s PBS, 2 packs
• The antibody-coupled support retains the antibody during (Coupling Buffer and Co-IP Buffer)
elution of the co-IP complex, simplifying analysis (1 L total volume) 60 ml
• Detection of the co-precipitated products by SDS-PAGE is Quenching Buffer 60 ml
Wash Solution 0.5 ml
accomplished without interference from antibody fragments Sodium Cyanoborohydride Solution (5 M) 50 ea.
• Covalent attachment of antibody conserves costly antibody Pierce Spin Cups 144 ea.
Pierce Microcentrifuge Tubes, 1.5 ml 50 ml
and allows the matrix to be used repeatedly IgG Elution Buffer
Lane Marker 5 ml
Labeled Proteins CoIP Sample Buffer, Non-reducing (5X)
MDM2 MDM2 23605 Pierce Mammalian Co-IP Kit Kit

MDM2 p53 Luc +p53 +Luc Sufficient material to immobilize up to 10 antibodies
and perform a minimum of 40 co-IP reactions if 25 µl
— MDM2 of antibody immobilized support is used. More
co-IPs can be performed if smaller volumes are used.
— Luciferase Includes same components as Product # 23600
and also includes:
M-PER Lysis Buffer 25 ml
— p53
Co-IP of p53 and MDM2 with mouse MAb to MDM2 using components of the
Thermo Scientific Pierce Co-Immunoprecipitation Kit. Purified mouse anti-
MDM2 was coupled to Thermo Scientific AminoLink Plus Coupling Resin.
Luciferase, MDM2 and p53 were translated and 35S-labeled. In vitro translated
p53 (5 µl) and MDM2 (5 µl) were combined and incubated at 30°C for 30 min-
utes. Co-IP was performed at 4°C for 2 hours with 60 µl anti-MDM2 antibody-
coupled resin. Luciferase was used as a negative control protein to incubate
with MDM2. Eluted proteins were resolved by 4–20% SDS-PAGE and visualized
by autoradiography.
Pierce HA and c-Myc IP/Co-IP Kits
sate
53 Ly
trol
rol
sate
Confirm two-hybrid interactions.
Cont
. Con
yc-p
Ly
Neg.
c-M
Co-I
Neg
LTA
The Thermo Scientific Pierce HA and c-Myc Tagged Protein Co-IP
IP
Kits include high-affinity, high-specificity antibodies coupled to 1 2 3 4 5 6
agarose to enable capture of HA- or c-Myc-tagged proteins along
with their binding partners. The covalent linkage between antibody
and the resin ensures results free from antibody contamination — LTA
and with minimal background. The mammalian kits also include
M-PER Mammalian Protein Extraction Reagent, which quickly and
gently lyses mammalian cells for easy preparation of extracts that
are compatible with co-immunoprecipitation (co-IP).
— c-Myc-p53
Highlights:
• Specific, high-affinity antibody provides high yield and
minimal background Co-IP of SV40 large T-antigen (LTA) with c-Myc-tagged p53. LTA and
• No antibody contamination in eluted sample c-Myc-p53 were expressed 35S-labeled in vitro (Lane 1 and 2). Before IP
• Control agarose resin included to test nonspecific binding or and co-IP, the lysates (5 µl of each) of LTA and c-Myc-p53 (Lanes 3 and 6),
c-Myc-p53 alone (Lane 4) or LTA alone (Lane 5) were incubated at 30˚C for
pre-clear sample
1 hour. Anti-c-Myc agarose (5 µg antibody in 10 µl slurry) (Lanes 3, 4 and 5) or
• Control tagged protein included to verify kit performance plain agarose slurry (Lane 6) was added to the corresponding sample. IP and
• Complete kits for IP or co-IP, with no reagent formulation necessary co-IP reactions were performed at 4˚C overnight. IP and co-IP products were
eluted and separated by 12% SDS-PAGE. The 35S-labeled proteins were
• Scalable for different antibody-resin and lysate requirements detected by fluorography.
• Includes Mini-Spin Columns for efficient washing and elution steps
IP IP Ordering Information
e
e
Pierc
Pierc

tific
tific
23610 Pierce HA-Tag IP/Co-IP Kit Kit

sate
cien
cien
ker
ker
lier A
lier B
lier A
lier B
lier D
lier C
Supp r C
Sufficient materials for 25 IP/co-IP assays with HA-

e
yc Ly
mo S
mo S
Mar
Mar
ysat
lie
tagged proteins.
Supp
Supp
Supp
Supp
Supp
Supp
HA L
Includes: Anti-HA Antibody Agarose Resin

Ther
Ther
150 µl
MW
MW
c-M
Tris Buffered Saline Pack 1 pack

Elution Buffer, pH 2.8 50 ml
Sample Loading Buffer (5X) 5 ml
Pierce Spin Cups 27/pkg
Pierce Microcentrifuge Tubes 100/pkg
HA-Tagged Positive Control 500 µl
23615 Pierce Mammalian HA-Tag IP/Co-IP Kit Kit

GST-HA — — GST-c-Myc
and also includes:
Extraction Reagent
23620 Pierce c-Myc-Tag IP/Co-IP Kit Kit

Sufficient materials for 25 IP/co-IP assays with
Comparison of the effectiveness of anti-HA- and anti-c-Myc-coupled resins. c-Myc-tagged proteins.
Thermo Scientific Pierce Anti-HA and anti-c-Myc resins show high affinity and Includes: Anti-HA Antibody Agarose Resin 250 µl
high specificity. Each IP was conducted with the same amount of antibody Tris Buffered Saline Pack 1 pack
(10 µg of immobilized HA antibody or 5 µg of immobilized c-Myc antibody) and Elution Buffer, pH 2.8 50 ml
the same amount of GST-HA or GST-c-Myc tag containing lysate (50 µl). Sample Loading Buffer (5X) 5 ml
Pierce Spin Cups 27/pkg
Pierce Microcentrifuge Tubes 100/pkg
c-Myc-Tagged Positive Control 500 µl
23625 Pierce c-Myc-Tag IP/Co-IP Kit Kit

and also includes:
Extraction Reagentt

Pierce Plus IgG Orientation Kits Thermo Scientific Pierce Protein G IgG Plus Orientation kit schematic
Greater antibody-binding capacities than Classic IgG Orientation DSS

Kits; available with either recombinant Protein A or Protein G.
Agarose
Agarose
Crosslinker
Bead
Bead
Protein Protein
G G
Thermo Scientific Pierce Plus IgG Orientation Kits provide for
efficient binding and covalent crosslinking of antibodies to Thermo
Scientific Pierce Protein A and Protein G Agarose Resin for use
References
in affinity column purification of antigens. The method improves Venturi, M., et al. (2000). J. Biol. Chem. 275(7), 4734–4742.
upon the traditional orientation mechanism by replacing the use of Sharp, D.A., et al. (2005). J. Biol. Chem. 280, 19401–19409.
dimethyl pimelimidate (DMP) with disuccinimidyl suberate (DSS) Liang, T.W., et al. (2002). J. Immunol. 168, 1618–1626.
for the crosslinking step. Our high-quality Protein A and Protein
G Agarose Resin provides for efficient antibody binding and
chromatography, and the DSS provides for simple and efficient Ordering Information
crosslinking of the bound antibody. The result is an affinity column
that can be used multiple times to purify the specific antigen of the Product # Description Pkg. Size
immobilized antibody from cell lysates and other samples. 44893 Pierce Protein A IgG Plus Orientation Kit Kit
Sufficient reagents for preparing 2 x 2 ml columns.
Highlights: Maximum recommended loading capacity:
16 mg Rabbit IgG per column
• DSS crosslinking system allows for higher antibody loading and Includes: Recombinant Protein A Agarose Columns 2 x 2 ml
less antibody leaching than imidoesters like DMP DSS Crosslinker 2 x 13 mg
Phosphate Buffered Saline 1/pkg
• Allows the positioning of IgG with the antigen-binding sites Blocking Buffer 6 ml
directed outward, capturing passing antigen in the mobile phase Elution Buffer 15 ml
Binding/Wash Buffer 120 ml
• Complete kits include all the reagents needed to immobilize Column Accessories
the antibody
44990 Pierce Protein G IgG Plus Orientation Kit Kit
Sufficient reagents for preparing 2 x 2 ml columns.
Maximum recommended loading capacity:
16 mg Rabbit IgG per column
Includes: Recombinant Protein G Agarose Columns 2 x 2 ml
DSS Crosslinker 2 x 13 mg
Phosphate Buffered Saline 1/pkg
Blocking Buffer 6 ml
Binding/Wash Buffer 120 ml
Column Accessories
Affinity Based Contaminant Removal
Removal of Detergent
However necessary the use of detergent may have been for initial
cell lysis or membrane protein extractions, subsequent applications
or experiments with the extracted proteins often require removal
of some or all of the detergent. For example, although many water-
soluble proteins are functional in detergent-solubilized form,
membrane proteins are often modified and inactivated by detergent
solubilization as a result of native lipid interactions having been
disrupted. In some such cases, membrane protein function is
restored when they are reconstituted into bilayer membranes by
replacement of detergent with phospholipids or other membrane-
like lipid mixtures.
The function of an individual protein can be studied in isolation if it

is first purified and then reconstituted into an artificial membrane
(although recovery of native orientation in the membrane is a
major challenge). Even when restoration of protein function is
not an issue, detergent concentration might have to be decreased
Contaminant Removal in a sample to make it compatible with protein assays or gel
electrophoresis.
Following the primary purification procedure to obtain a sample
of interest, secondary purification steps to remove contaminants Detergent removal can be attempted in a number of ways.
may be required. The contaminants might be inhibitors, interfering Dialysis is effective for removal of detergents that have high CMCs
substances or inappropriate buffers. A number of available affinity and/or small aggregation numbers, such the N-octyl glucosides.
supports allow researchers to either specifically purify a protein Detergents with low CMCs and large aggregation numbers
of interest away from a complex mixture of biological molecules cannot be dialyzed because most of the detergent molecules exist
(positive selection) or remove specific contaminants from a sample in micelles that are too large to diffuse through the pores of the
containing a protein of interest (negative selection). Nearly any dialysis membrane; only excess monomer can be dialyzed. Ion
given affinity purification system can be used for either positive or exchange chromatography using appropriate conditions to
negative selection, depending on whether the non-bound or eluted selectively bind and elute the proteins of interest is another
fraction is recovered. For example, immobilized Protein A can be effective way to remove detergent. Sucrose density gradient
used for general affinity purification of antibodies (positive selection), separation also can be used.
but it can also be used to selectively remove immunoglobulins from
a sample in which they are considered a contaminant (negative Thermo Scientific Detergent Removal Gel allows selective
selection). Following is a brief description of affinity-based affinity-based removal of many different detergents from solutions.
systems for removal of contaminants by negative selection.

Thermo Scientific Products for Affinity Based
Contaminant Removal
Detergent binding data.

Detergent Removing Gel
Capacity
Makes detergent removal efficient, fast and easy with high protein Detergent Product # (mg/ml gel) Binding Conditions
recoveries, too. ®
Brij -35 28316 80 100 mM Phosphate Buffer, pH 7.0
Highlights: CHAPS 28300 50 50 mM Tris Buffer, pH 9.0
• Allows relatively small detergent molecules to enter the gel SDS 28312 92 50 mM Tris Buffer, pH 9.0
matrix where they interact with a specially developed ligand
Triton® X-100 28314 57 100 mM Phosphate Buffer, pH 7.0
capable of removing them from solution
Tween® -20 28320 74 100 mM Phosphate Buffer, pH 7.0
• Low exclusion limit of the support overcomes nonspecific binding
• For use with biological macromolecules greater than 10 kDa References
1. Bubien, J.K., et al. (2001). J. Biol. Chem. 276, 8557–8566.
• Detergent is extracted without loss of valuable protein 2. Carman, C.V., et al. (2000). J. Biol. Chem. 275, 10443–10452.
• Reusable affinity matrix can be regenerated up to three times 3. Rouse J.C. and Vath J.E. (1996). Anal Biochem. 238, 82–92.
4. Gentile, F., et al. (1997). J. Biol. Chem. 272, 639–646.
• Compatible with a wide variety of buffers, pH 3.5–10
• Recovery of dilute protein solutions enhanced by use of a
carrier protein
Applications:
20208 Detergent Removing Gel 10 ml
• Reconstitution of proteoliposomes to study integral membrane
proteins1,2 20303 Detergent Removing Gel 10 x 10 ml
• Preparation of samples for mass spectroscopy3,4 20346 Detergent Removing Gel 5 x 1 ml
pre-packed
columns
Thermo Scientific Products for Affinity Based
Contaminant Removal
Removal of Albumin Albumin Albumin

Contamination Depleted
Human serum albumin (HSA) can account for greater than 60% of
the total protein in serum samples and can have a concentration
of ~40 mg/ml. This high concentration of albumin frequently inter-
feres with analysis of proteins of biological interest. Traditionally,
researchers have produced albumin-free samples using chromato-
graphic methods involving multiple purification steps. The Thermo
Scientific Pierce Blue Albumin Removal Kit takes advantage of the A B
albumin-binding properties of immobilized Cibacron® Blue F3GA
Dye and is designed for rapid treatment of small sample volumes Thermo Scientific Pierce Blue Albumin Removal Kit for 2-D gel analysis.
commonly used in proteomic studies. Serum sample obtained by diluting 10 µl human serum with 40 µl TBS (Product
# 28376) and loading 5 µl onto Gel A. Albumin-free sample obtained by diluting
50 µl human serum 1:1 with buffer, adding to a Pierce Blue Disc, washing the
resin three times with 50 µl buffer, combining the fractions, and loading 5 µl
Pierce Blue Albumin Removal Kit onto Gel B. Both samples were focused using pH 4–7 isoelectric focusing (IEF)
strips and run on 8–16% Tris-glycine gels.
Albumin-free serum samples in less than 15 minutes.
The Thermo Scientific Pierce Blue Albumin Removal Kit is Ordering Information
designed to remove albumin from human serum quickly and
consistently while achieving higher protein yields. Product # Description Pkg. Size
89845 Pierce Blue Albumin Removal Kit† Kit
Each Blue Disc has the capacity (≥ 2 mg HSA) to remove the Sufficient reagents for 12–25 reactions.
majority of albumin from 10–150 µl of serum in less than 15 min- Includes: Pierce Blue Albumin Removal Discs 25 discs
Binding/Wash Buffer 6.25 ml
utes. The kit procedure has been optimized for removal of human Pierce Spin Columns 25 columns
serum albumin but will also effectively remove swine and sheep
serum albumin. With a slight modification of the binding buffer, † See patent information on inside back cover.
the Blue Albumin Removal Kit can also be used to remove excess
bovine, calf, goat and rat albumin. The product, however, is not
recommended for mouse albumin.
Our Blue Albumin Removal Buffer is compatible with most down-

stream applications and does not interfere with protein assays or
introduce contaminants for SDS-PAGE. Simply add the albumin-
depleted serum to the appropriate sample buffer and the sample is
ready for any application including 2-D gel applications.
SERUM FILTRATE BUFFER
Step 1. Step 2. Step 3. Step 4. Step 5.

Hydrate Thermo Scientific Add serum sample to the resin. Recover filtrate and add to the resin. Add Binding/Elution Buffer. Combine desired fractions.
Pierce Disc in water. Spin. Incubate 2 minutes. Incubate 2 minutes and spin. Spin. Wash 1-4 times. Concentrate if needed.
Total Time: 10-15 minutes
Thermo Scientific Pierce Albumin Removal Kit protocol.

Removal of Endotoxin Removal of Nonspecific Antibodies
Endotoxins are pyrogenic lipopolysaccharide (LPS) components Because they contain mixtures of immunoglobulins, preparations
of gram-negative bacteria. Eliminating endotoxin contamination of polyclonal antibodies from serum or other samples often have
in aqueous and physiological solutions is a difficult and often cross-reactivity to unintended targets in the sample being probed
expensive process. Complete removal of all contaminating pyrogens in Western blot or ELISA. It is important to purchase or prepare
is not feasible; the most likely outcome is a reduction of endotoxins antibodies that do not cross-react in this way. For example, by
to tolerable levels for a given study system. Typically, a pyrogen- passing a rabbit anti-mouse IgG polyclonal antibody sample over
poor (i.e., safe for use) preparation of protein will contain less than a column of immobilized human serum proteins, one can ensure
1.0 endotoxin unit (EU) per milligram of protein. that the resulting antibody will react only to mouse IgG primary
antibody without cross-reacting with human immunoglobulins
Thermo Scientific Detoxi-Gel Endotoxin Removing Gel uses immo- in the sample. We offer selected antibodies that have been pre-
bilized polymixin B to bind and remove endotoxins from solutions. adsorbed in this way to prevent cross-reactivity to unintended
The product is commonly used to remove endotoxins from protein immunoglobulin species. Such pre-adsorption of antibodies is
solutions, cell culture media, solutions containing pharmacological a form of negative selection affinity purification wherein the
components and aqueous buffers. immobilized ligand is a mixture of proteins.
Certain substances and proteins in solution bind strongly to Cross-reactivity of antibodies to bacterial proteins is a common
endotoxin, thereby either decreasing binding capacity of Detoxi- problem for researchers investigating recombinant proteins
Gel Support or resulting in loss of protein from the sample (i.e., prepared in Escherichia coli. The possibility of such cross-
the protein remains bound to endotoxin when the endotoxin binds reactivity can be reduced or eliminated by removing immunoglobulins
to the immobilized polymixin B). This is especially true of proteins in the polyclonal antibody sample that bind to proteins from
like bovine serum albumin (BSA) and ovalbumin. BSA is a common E. coli. We offer the Thermo Scientific Immobilized E. coli Lysate Kit
component of tissue media and, as such, it is also often a source for this purpose. Proteins from total lysate of E. coli strain BMH 71–18
of endotoxin contamination. have been immobilized onto a crosslinked 4% agarose support.
Detoxi-Gel Endotoxin Removing Gel Immobilized E. coli Lysate and Kit

Eliminate worries about endotoxins interfering with your test results. For clean, easy removal of E. coli-reactive antibodies.
Highlights: High background, or low signal:noise ratio, is often a problem
• Uses immobilized polymixin B to remove endotoxins by binding when screening libraries. Crude antisera and ascites fluid often
to their lipid A domains contain IgG components that bind to Escherichia coli proteins. This
• 1 ml of Thermo Scientific Detoxi-Gel Resin removes at least 9,995 can be especially problematic if the titer or binding affinities of the
endotoxin units from a 5 ml sample containing 10,000 EU of lipopoly- E. coli binding antibodies are higher than that of the antibody to the
saccharide (LPS) protein of interest, resulting in false positives.
• Reusable – no loss in binding capacity even after 10 regenerations
To make removal of E. coli antibodies cleaner and easier, we
• Requires activation and regeneration with 1% sodium deoxycholate
immobilized E. coli proteins on a solid support. When a sample is
• Protein recovery dependent upon protein type and concentration
passed over the column, the purified antibody is collected in the
– empirical testing required
void volume. The contaminating E. coli antibodies are adsorbed
References
onto the matrix, and the purified antisera is collected.
Chigaev, A., et al. (2003). J. Biol. Chem. 278, 38174–38182.
Gao, B. and Tsan, M. (2003). J. Biol. Chem. 278, 22523–22529.
Hahn-Dantona, E., et al. (2001). J. Biol. Chem. 276, 15498–15503.
Zhang, J., et al. (2000). FASEB J. 14, 2589–2600.
Ordering Information 44938 Immobilized E. coli Lysate 5 ml

E. coli proteins from strain BMH 71–18
immobilized on crosslinked 4% beaded agarose.
44940 Immobilized E. coli Lysate Kit Kit
20344 Detoxi-Gel Prepacked Columns 5 x 1 ml Includes: Prepacked Column of Immobilized 2 ml
E. coli Lysate
20339 Detoxi-Gel Endotoxin Removing Gel 10 ml BupH Tris Buffered Saline 500 ml
Regeneration Buffer 250 ml
20340 Detoxi-Gel Endotoxin Removing Gel 1,000 ml Column Extender
Additional Thermo Scientific Affinity Supports
Immobilized Soybean Trypsin Inhibitor

For effective removal of trypsin, chymotrypsin and elastase from
protein digests.
Applications:
• Purifying trypsin, chymotrypsin and elastase1,2
• Removing proteases from activated pancreatic juices3
References
1. Feinstein, G., et al. (1974). Euro. J. Biochem. 43(3), 569–581.
2. Peterson, L.M., et al. (1976). Biochemistry 15(12), 2501–2508.
3. Reeck, G.R., et al. (1971). Biochemistry 10(25), 4690–4698.

20235 Immobilized Soybean Trypsin Inhibitor 2 ml
Support: 4% beaded agarose
Capacity: ≥ 6 mg trypsin/ml of resin
In addition to the few affinity supports whose ligands have broad
application to many different protein methods, there are many
others whose applications are more narrowly defined or are in-
corporated into kits for very specific purposes. These include kits Immobilized Pepstatin
to isolate cell surface proteins using biotinylation and NeutrAvidin
An excellent cathepsin binding matrix.
Agarose, kits to enrich for phosphoproteins, glycoproteins and
ubiquitinated proteins. The stand-alone resins on this page and the
next include those that can be used to remove trypsin from protein H H H
Agarose
N N N
Bead
digest, isolate blood proteins, purify C-reactive protein and capture Pepstatin
ribonucleosides. In addition, page 74 presents our complete line of
magnetic beads, together with their magnet accessories.
Pepstatin = R Ala R Val Val i-Val
R = (4-amino-3-hydroxy-6-methyl) heptanoic acid
Thermo Scientific Immobilized Pepstatin
Reference
Helseth, Jr., D.L. and Veis, A. (1984). Proc. Natl. Acad. Sci. USA 81, 3302–3306.

20215 Immobilized Pepstatin 5 ml
Spacer: Diaminodipropylamine
Capacity: 1–2 mg of pepsin/ml of resin

Additional Thermo Scientific Affinity Supports and Kits
Immobilized Heparin Immobilized D-Galactose

Use to isolate many blood proteins that have enzymatic activities. For lectin and galactosidase binding.
Applications: Applications:
• Enrich lysates for nucleic acid-binding proteins • Human alpha-galactosidase A purification1
• Isolate many blood proteins • E. coli heat labile enterotoxin purification2
• C-type lectin purification3
Reference
Smith, P.K., et al. (1980). Anal. Biochem. 109, 466–473. • Cholera toxin (CT) purification4,5
OH
O OH
CH2OSO3- O O
Agarose
O
Bead
O S
Agarose
COO- HO
Bead
O O O O
OH OH OH
O
OSO3- NHOSO3-
Thermo Scientific Immobilized Thio-alpha-D-Galactose
n
References
Thermo Scientific Immobilized Heparin 1. Yasuda, K., et al. (2004). Protein Expression and Purification. 37, 499–506.
2. Okamoto, K., et al. (1998). J. Bacteriol. 180, 1368–1374.
3. Matsumoto, J., et al. (2001). Development. 128, 3339–3347.
Ordering Information 4. Bowman, C.C. and Clements, J.D., et al. (2001). Infec. Immun. 69, 1528–1535.
5. Tinker, J.K., et al. (2003). Infec. Immun. 71, 4093–4101.
20207 Immobilized Heparin Kit Ordering Information
Loading: ≥ 0.2 mg of heparin/ml of resin
(determined by the colorimetric method)
20372 Immobilized D-Galactose 5 ml
Immobilized p-Aminophenyl Phosphoryl Choline Capacity: ≥ 20 mg jacalin/ml resin
For C-reactive protein binding.

Immobilized Boronic Acid
HN O For ribonucleoside isolation.
Agarose
O
Bead
P N+
O OH
O-
Resin
Bead
O H
N B OH
OH
Thermo Scientific Immobilized p-Aminophenyl Phosphoryl Choline
O
Reference
Robey, F.A. and Liu, T.Y. (1981). J. Biol. Chem. 256, 969–975.
Thermo Scientific Immobilized Boronic Acid
Ordering Information References

Vlassara, H., et al. (1981). Proc. Natl. Acad. Sci. USA 78, 5190–5192.
Product # Description Pkg. Size Gehrke, C.W., et al. (1978). J. Chromatogr. 150, 455–476.
20307 Immobilized p-Aminophenyl Phosphoryl 5 ml

Choline Ordering Information
Capacity: ≥ 3 mg of human C-reactive protein/ml of resin
20244 Immobilized Boronic Acid 10 ml
Support: Polyacrylamide resin beads
Capacity: ≥ 99% binding and recovery of 110 µmol
AMP/ml of resin
Spacer: m-aminophenyl group
Loading: 100 µmol boronate/ml of resin
Pierce Cell Surface Protein Isolation Kit How it works

The cell surface protein isolation procedure uses a cell-imperme-
Convenient biotinylation and isolation of cell surface proteins for able, cleavable biotinylation reagent to label surface proteins at
Western blot analysis. exposed primary amines. Cells are then harvested and lysed, and
the labeled surface proteins are affinity-purified using Thermo
The Thermo Scientific Cell Surface Protein Isolation Kit is a Scientific NeutrAvidin Agarose Resin (see Figure below).
complete kit for the convenient biotinylation and isolation of
mammalian cell surface proteins, specifically targeting cell The biotinylation reagent, Sulfo-NHS-SS-Biotin, does not permeate
surface proteins to the exclusion of intracellular proteins. intact cell membranes so labeling occurs only on proteins exposed
The kit efficiently labels proteins with accessible lysine to the cell surface. Labeling occurs at primary amines (-NH2
residues and sufficient extracellular exposure. groups), which exist in proteins at the N-terminus and side chain
of lysine residues. After cell lysis, the cell surface proteins that
Highlights: were biotinylated by Sulfo-NHS-SS-Biotin are captured by its high-
• Isolates cell surface proteins – reduces complexity of total affinity interaction with NeutrAvidin Agarose Resin. While surface
cellular protein protein are bound to the resin beads, all other proteins and cellular
• Efficiently recovers labeled proteins – cleavable biotin allows components in the lysate are washed away using the convenient
for nearly 100% recovery of isolated cell surface proteins microcentrifuge spin columns supplied in the kit. Finally, because
• Convenience – all reagents are provided in one kit, along with Sulfo-NHS-SS-Biotin contains a disulfide bond (-S-S-) in its spacer
complete instructions for labeling, cell lysis and purification of arm, the labeled cell surface proteins are efficiently recovered from
cell surface proteins the resin by cleaving the disulfide bond with SDS-PAGE sample
• Western blotting applications – proteins recovered in SDS-PAGE loading buffer that contains dithiothreitol (DTT). The isolated cell
buffer are loaded directly onto polyacrylamide gels surface proteins contain a small, nonreactive tag of the originally
• Robust system – protocol designed for diverse cell lines labeled primary amines but are no longer biotinylated (biotin
remains bound to the resin).
Isolate biotinylated proteins

Transfer cell pellet on Thermo Scientific
45
Biotinylate cells Quench Reaction 40 to 1.5 ml tube NeutrAvidin Resin
35
30
25
20
30 minutes at 4°C Harvest Cells 15

10
Lyse cells
75
50 30 minutes on ice
N
1-D gel
bead N Wash gel then elute with SDS-PAGE bead N
sample buffer + 50 mM DTT
Perform
B electrophoresis
+ or other application
N SH
protein – SH
B
S-S-protein
Thermo Scientific NeutrAvidin B Biotin

N Biotin-Binding Protein
Procedure for the Thermo Scientific Pierce Cell Surface Protein Isolation Kit.

A. Cell Surface Proteins
+ - + -
——— ——— ——— ———
R F E R F E R F E R F E
89881 Pierce Cell Surface Protein Kit
EGFR Integrin α5 Isolation Kit
+ - + - Includes: EZ-Link Sulfo-NHS-SS-Biotin 8 x 12 mg vials
——— ——— ——— ———
R F E R F E R F E R F E Quenching Solution 16 ml
Lysis Buffer 4.5 ml
NeutrAvidin Agarose Resin 2.25 ml
Wash Buffer 34 ml
EIGF-1Rβ Integrin β1 Spin Columns and Accessories 1 pack
Dithiothreitol (DTT) 8 x 7.7 mg microtubes
Phosphate Buffered Saline 2 packs
B. Intracellular Proteins
Tris Buffered Saline 1 pack
+ - + -
——— ——— ——— ———
R F E R F E R F E R F E
Hsp90 Calnexin
Protein isolation is specific to cell surface proteins. Panels are Western blot
results for known cell surface proteins (Panel A) and intracellular proteins
(Panel B) from HeLa cells tested with the Thermo Scientific Pierce Cell Surface
Protein Isolation Kit. Plus symbol (+) denotes results for cells treated with the
Sulfo-NHS-SS-Biotin reagent; minus symbol (-) denotes results for cells that
were not treated with the biotin reagent but were otherwise carried through
the kit procedure. Lanes are no-sample resin-control (R), flow-through (F) and
eluted (E) fractions. Presence of target cell surface proteins in the plus-E and
minus-F conditions indicate successful isolation with the kit. Presence of intra-
cellular proteins in F condition of both plus and minus conditions indicates that
the labeling and purification procedure is specific to cell surface proteins.
Differential expression of cell surface proteins in response to EGF. A431 and

HeLa cells were treated with or without 20 ng/ml and 10 ng/ml EGF for 16 hours,
respectively. Both cell types were processed with the Thermo Scientific Pierce Cell
Surface Protein Isolation Kit protocol. Elution fractions were analyzed by Western
A. A431 Cells B. HeLa Cells
EGF - - + + EGF - - + +
Integrin α5 EGFR
- - + +
Integrin β1
blot for the quantities of A. integrin β1 and integrin α5 subunits or B. EGFR.
Pierce Phosphoprotein Enrichment Kit FT W E L
Enrich phosphoproteins for analysis in Western blotting or Phospho-MAPK T202/Y204

mass spectrometry.
The Thermo Scientific Pierce Phosphoprotein Enrichment Kit is Phospho-Akt S473

designed for the efficient enrichment of phosphorylated proteins
derived from mammalian cells and tissues. The proprietary resin
and buffer composition produces superior yields with negligible Phospho-Src Y527
non-specific binding. The kit is offered in a convenient spin format
and is compatible with downstream applications including
Western blotting, Mass Spec, 2D-PAGE, and protein arrays. Cytochrome C
Highlights:
p15Ink4b
• Specific – low nonspecific protein contamination from complex
biological samples, such as cell culture lysate and mouse
tissue extract
• Fast – easy spin format enables enrichment of phosphorylated Enrich complex biological samples for phosphorylated proteins with high
specificity. Western blotting analysis reveals a stronger signal for the elution
proteins in less than 2 hours fraction (E) bands (processed sample) than the total cell extract (L) bands
• Superior Yield – achieves higher yields than other commercially (unprocessed sample) for all phosphoproteins tested. Furthermore, the
available kits (see Table) non-phosphorylated proteins cytochrome c and p15Ink4b are negative controls
and demonstrate the specificity of the kit. Total cell extract (2 mg) from serum-
• Convenient format – complete kit offering pre-dispensed spin starved NIH 3T3 cells was used for each enrichment procedure. Ten µg of
columns, buffers, reagents and ultrafiltration columns for total protein was loaded in each lane and Western blotting was performed
enrichment using antibodies that detect site-specific phosphorylation. FT: flow-through
• Compatible – downstream applications include mass spectrometry, and W: wash.
Western blotting, and 2D-PAGE
The Thermo Scientific Pierce Phosphoprotein Enrichment Kit provides higher Product # Description Pkg. Size
phosphoprotein yields in less time than other kits. 90003 Pierce Phosphoprotein Enrichment Kit Kit
Includes: Phosphoprotein Enrichment Columns 10 ea.
Phosphoprotein Enrichment Time (1 ml resin bed)
Kit Yield (µg)† (Hours) Lysis/Binding/Wash Buffer 325 ml
Our Phosphoprotein Enrichment Kit 300 1.5 CHAPS 1g
Protein Concentrators (7ml/9K MWCO) 10 ea.
Supplier Q Kit 88 4.5
White Column Caps 10 ea.
Supplier I Kit 52‡ 3.5
Supplier C Kit 160 3
Supplier E Kit Too dilute to 5
determine
† From 2 mg total protein.

‡ Based on maximum 1 mg load per manufacturer’s protocol.

Human Serum CHO Lysate
Pierce ConA and WGA Glycoprotein Isolation Kits Glycoprotein isolation from
human serum and cell lysate:
Supplier A
Supplier G
Supplier A
Supplier G
Pierce Kit
Pierce Kit
Isolate glycoproteins from complex protein mixtures. performance comparison of
ConA
ConA
kits using ConA resin. Human
serum and CHO lysate samples
Glycosylation is a post-translational modification that plays an were processed with the
important role in biological functions, including immune regulation, Thermo Scientific Pierce
inflammation, cell-to-cell adhesion, and cell signaling. We offer Glycoprotein Isolation Kit and
two lectin-based glycoprotein isolation kits, concanavalin A (ConA) other commercially available
and wheat germ agglutinin (WGA), that allow isolation of glycopro- ConA resins. An equivalent
amount of total protein was
teins from complex protein mixtures, including serum, tissue applied to each resin. Eluted
and cultured cell lysates, thus enabling downstream analysis. ConA glycoprotein fractions were
lectin recognizes α-linked mannose and terminal glucose residues, compared with ConA Resin
while WGA lectin selectively binds to N-Acetyl glucosamine boiled in SDS-PAGE Buffer to
release lectins. All fractions were
(GlcNAc) groups and to sialic acid. normalized by volume, resolved
on 8–16% polyacrylamide gels
Highlights: alongside purified ConA
• High recovery – equivalent or greater glycoprotein recovery (rightmost lanes) then silver-stained. Arrows identify protein bands that result from
vs. competitor kits and lectin resins ConA leaching from the resin during the elution process.
• Fast – glycoprotein purification in less than one hour
• Versatile – isolate glycoproteins from various sample types; Ordering Information
e.g., human serum and cell lysate
• Robust – lectin does not leach from resin when processing sample Product # Description Pkg. Size
• Convenient – complete kit offering lectin resins and spin columns 89804 Pierce Glycoprotein Isolation Kit, ConA Kit
with all necessary reagents Sufficient reagents to isolate glycoproteins with
strong affinity for ConA from 10 samples of up to
• Compatible with Bradford-based protein assays – dialysis or 640 µl (1-1.5 mg total protein) each.
protein precipitation of recovered glycoproteins is not required Includes: ConA Lectin Resin 1.1 ml
prior to protein assay Binding/Wash Buffer, 5X Stock Solution 6.5 ml
Elution Buffer 5 ml
Glycoprotein isolation from human serum and Spin Columns and Accessories 1 pack
Human Serum CHO Lysate
cell lysate: performance comparison of kits 89805 Pierce Glycoprotein Isolation Kit, WGA Kit
Supplier A
Supplier A
using WGA resin. Human serum and CHO lysate

Pierce Kit
Pierce Kit
Sufficient reagents to isolate glycoproteins with

samples were processed with the Thermo strong affinity for WGA from 10 samples of up to
Scientific Pierce Glycoprotein Isolation Kit, WGA 640 µl (1–1.5 mg total protein) each.
and with another commercially available WGA Includes: WGA Lectin Resin 1.1 ml
resin. An equivalent amount of total protein was Binding/Wash Buffer, 5X Stock Solution 6.5 ml
applied to each resin. Eluted glycoprotein frac- Elution Buffer 5 ml
Spin Columns and Accessories 1 pack
tions were normalized by volume, resolved on 8–
16% polyacrylamide gels and silver stained.
1. Pipette 200 µl of 2. Wash resin 3X 3. Dilute glycoprotein 4. Add diluted glycoprotein 5. Centrifuge 6. Wash resin 7. Add 200 µl of 8. Centrifuge.
resin slurry to spin with 200 µl of sample in sample to column and and discard with 400 µl of Elution Buffer Collect eluate
column. Centrifuge Binding/Wash Binding/Wash mix for 10 minutes. flow-through. Binding/Wash and mix for and repeat
column to remove Buffer. Buffer (4:1). Buffer 4X. 5 10 minutes. steps 7 and 8.
storage buffer.
Thermo Scientific Pierce Glycoprotein Isolation Kit protocol.
Pierce Ubiquitin Enrichment Kit involved in a variety of cellular processes, including DNA repair,
transcriptional regulation, signal transduction, cell metabolism
Capture more ubiquitin-modified proteins! and morphogenesis. Differences in total ubiquitination or the
ubiquitination of specific proteins affect numerous pathological
The Thermo Scientific Pierce Ubiquitin Enrichment Kit facilitates the conditions, including malignancies, certain genetic diseases and
isolation of polyubiquitin protein conjugates from cultured cells and neurodegenerative diseases.
tissue samples. The enriched fraction can subsequently be analyzed
to determine the amount of general ubiquitin conjugates present Thermo
Scientific Supplier Ab-based GSH
or to identify a specific protein of interest by Western blotting. The Kit C Method Resin
assay protocol is fast and straightforward, allowing for isolation
H F E F E F E F E
of polyubiquitinated proteins and the fractionation of monoubiqui-
kDa
tinated species in the resin flow-through. The Ubiquitin Enrichment
Kit outperforms kits from other suppliers and provides a clean and 220
specific preparation of proteins when compared to a control resin. 120
100
Highlights: 80
• Fast – less than 45 minutes hands-on time 60

50
• Complete – includes all reagents needed for ubiquitin-modified
40
protein enrichment from cultured cells and tissue samples
• Flexible – sample incubation from 2 hours to overnight allows 20
assay to be completed in several hours or in less than 30 minutes
after overnight incubation
Thermo Scientific Pierce Ubiquitin Enrichment Kit recovers more ubiqui-
• Robust – compatible with all Pierce Cell Lysis Products and tin-modified proteins than any other available method. Western blot image
standard RIPA formulations reflects detection of samples using the anti-ubiquitin antibody supplied in the
• Multiple-sample format – easily processes 1–15 samples concurrently kit. Epoxomicin-treated HeLa cells lysates (150 µg) were processed by four
different methods. The resulting flow-through (F) and elution (F) fractions were
volume-normalized to the original unprocessed lysate (H) and identical volumes
Thermo Scientific Pierce Ubiquitin Enrichment Kit protocol. electophoresed for Western blot detection. Compared to Supplier C’s Kit and
an antibody-based method, the Pierce Kit yielded more ubiquitinated protein
The ubiquitin proteasome pathway is the principal non-lysosomal in the elution fraction (and less protein in the flow-through fraction), indicating
pathway that controls the proteolysis. This pathway is significantly significantly better enrichment of ubquitinated proteins. GSH Resin is a negative
control for comparison.
Cell or tissue lysate
150 µg (150 µl)
Add 150 µl sample Product # Description Pkg. Size

dilution buffer and 20 µl
ubiquitin affinity resin 89899 Pierce Ubiquitin Enrichment Kit Kit
Sufficient materials for enriching up to 15 lysate
samples containing ~0.15 mg total protein per sample.
Includes: Pack 1
Incubate at 4˚C for
2 hours or overnight Polyubiquitin Positive Control (1,000X) 50 µl
Anti-ubiquitin Antibody (rabbit antiserum) 50 µl
Pack 2
Flow-through Polyubiquitin Affinity Resin, supplied as 300 µl
Save for analysis
Centrifuge spin column a 25% slurry
at 5,000 x g for 15 seconds Resin Binding Capacity: ~1 µg per 20 µl of slurry
Tris Buffered Saline Pack 1 each
Spin Columns and Accessories 18 columns
Save washes Wash resin 3X

for analysis with wash buffer
Elute ubiquitinated protein

sample with SDS-PAGE
loading buffer or IEF buffer
Perform Western analysis using

anti-ubiquitin antisera (included with kit)
or antibody for your protein of interest

HisPur™ Cobalt Resin, Spin Columns, Co2
+
Ni2
+
Co2
+
Ni2
+
Co2
+
Ni2
+
and Chromatography Cartridges 1 2 3 4 5 6 L 1 2 3 4 5 6 L 1 2 3 4 5 6 L
Specific, fast and gentle purification of His-tagged proteins.
The preferred method for purifying recombinant His-tagged

proteins is immobilized metal affinity chromatography (IMAC).
Traditionally, chelating chromatography resins are charged with
either nickel or cobalt ions that coordinate with the histidine side
chains in the 6xHis-tag. HisPur Cobalt Resin is a tetradentate GFP β-gal Protein L
chelating resin charged with cobalt that binds His-tagged proteins
with high specificity and releases them under lower imidazole Thermo Scientific HisPur Cobalt Resin outperforms other IMAC resins. E. coli
concentrations than required with nickel resins (see Figure). lysates containing overexpressed His-tagged green fluorescent protein (GFP),
HisPur Cobalt Resin can be used to obtain high-purity proteins β-galactosidase or Protein L were applied to 200 µl bed volumes of HisPur
with no metal contamination. Cobalt Resin and several competing cobalt and nickel resins. The
first elution fraction for each IMAC resin was analyzed by SDS-PAGE and
protein purity determined. Gel lanes were normalized to equivalent volume.
The HisPur Chromatography Cartridges are convenient, reliable Lane 1 = HisPur Cobalt Resin, Lane 2 = supplier C’s cobalt resin, Lane 3 =
and ready-to-use pre-packed devices for the isolation of proteins supplier S’s cobalt resin, Lane 4 = supplier G’s nickel resin, Lane 5 = supplier
in solution and purification of His-tagged fusion proteins. The Q’s nickel resin, Lane 6 = Ni2+-IDA resin and Lane L = lysate load.
HisPur Cartridges are compatible with automated LC instrumen-
tation, such as the ÄKTÄ™ and BioLogic Systems, and adapt to Ordering Information
manual syringe processing.
Highlights:
• High purity – obtain pure protein without optimizing imidazole 89967 HisPur Cobalt Spin Columns, 0.2 ml 25 columns
washing conditions 89968 HisPur Cobalt Spin Columns, 1 ml 5 columns
• Specificity – cobalt:chelate binding core binds fewer contaminants, 89969 HisPur Cobalt Spin Columns, 3 ml 5 columns
resulting in lower background than nickel (see Table)
89964 HisPur Cobalt Resin 10 ml bottle
• Low metal leaching – no metal contamination in eluted sample
89965 HisPur Cobalt Resin 100 ml bottle
• Versatility – purify proteins under native or denaturing conditions;
compatible with cell lysis reagents and a variety of buffer additives 89966 HisPur Cobalt Resin 500 ml bottle
• Flexibility – available as bulk resin or predispensed columns 90090 HisPur Purification Kit, 0.2 ml 25 columns
compatible with both spin and gravity-flow formats 90091 HisPur Purification Kit, 1 ml 5 columns
• Cost effective – reuse or discard
90092 HisPur Purification Kit, 3 ml 5 columns
• Superior – performs better than other commercially available
IMAC Resins (see Figure) 90093 HisPur Chromatography Cartridges 5 x 1 ml
Wash Elution 90094 HisPur Chromatography Cartridges 2 x 5 ml
kDa M F 1 2 3 1 2 3 4 5 L
210
110
Thermo Scientific HisPur Cobalt Resin is specific for His-tagged proteins and
47 allows mild, efficient elutions. Bacterial lysate (1.0 mg total protein) was applied to
32 6xHis-GFP a 200 µl bed volume of HisPur Cobalt Resin in a spin column. Gel lanes were normal-
25 ized to equivalent volume. M = Molecular Weight Markers (Product # 26691), L =
16.5 lysate load and F = flow-through.
Thermo Scientific HisPur Cobalt Resin yields more His-tagged protein and higher purity than other Co2+ and Ni2+ IMAC Resins.
His-GFP His-β-Gal His-Protein L

Yield (µg)* Purity (%)** Yield (µg)* Purity (%)** Yield (µg)* Purity (%)**
Thermo Scientific HisPur Cobalt Resin 298 87 78 93 42 77
Supplier C Cobalt Resin 206 78 26 90 35 76
Supplier S Cobalt Resin 211 85 27 65 38 77
Supplier G Nickel Resin 239 84 42 83 29 68
Supplier Q Nickel Resin 242 85 24 48 30 72
Ni2+-IDA Resin 70 37 6 16 17 46
* Recovered from a 5 mg total protein load (total protein yield x purity).

** Purity of the first elution fraction.
MagnaBind Beads and Supports

A convenient method for isolating biomolecules using affinity
binding, while retaining biological activity.
Magnetic beads are a convenient affinity support for a variety

of assays, which allow easy purification of the target without
columns or centrifugation. After a binding step in an affinity
purification procedure, the magnetic particles are easily and
rapidly collected by placing the microcentrifuge tube or reaction
vessel next an appropriate rare-earth magnet (see Figure).
Thermo Scientific MagnaBind beads respond rapidly to
MagnaBind Magnets but can be easily dispersed and regathered
multiple times (i.e., they will not irreversibly aggregate) because
they do not have any magnetic memory. MagnaBind Beads are
available pre-coated with Protein A, Protein G, streptavidin, anti-
mouse or anti-rabbit antibodies. Activated beads, with either
amine or carboxyl groups, are also available for attaching other
proteins or affinity ligands to a magnetic particle.
Highlights:
• Available pre-coated with popular affinity ligands or derivatized
for covalent attachment of proteins and other specific ligands
• Beads do not irreversibly aggregate because they have no
magnetic memory; collect and disperse the beads multiple times
if needed
• Most separations require a short five- to 10-minute bench-top
procedure
Applications:
• Cell sorting using positive or negative selection
• Protein purification or immunoassays using direct or indirect methods Thermo Scientific MagnaBind Magnet for 1.5 ml Microcentrifuge Tube. Thermo
Scientific MagnaBind Beads in solutions within a microcentrifuge tube are rap-
Characteristics of underivatized Thermo Scientific MagnaBind Beads. idly “pelleted” when the tube is placed in the magnetized holder. Magnets for
six microcentrifuge tubes and 96-well microplates are also available.
Composition Silanized iron oxide
References
Magnetization 25–35 EMU/g Chaudhuri, T.K., et al. (2001). Cell 107, 235–246.
Type of Magnetization Superparamagnetic (no magnetic memory) Newey, S.E., et al. (2001). J. Biol. Chem. 276, 6645–6655.
Xu, X., et al. (2001). J. Biol. Chem. 276, 43221–43230.
Surface Area > 100 m2/g
Settling Rate 4% in 30 minutes Ordering Information
Effective Density 2.5 g/ml
Number of Beads 1 x 108 beads/mg
pH Stability Aqueous solution, above pH 4.0 21344 MagnaBind Streptavidin Beads 5 ml
Concentration 5 mg/ml 21348 MagnaBind Protein A Beads 5 ml

21349 MagnaBind Protein G Beads 5 ml
Note: To establish a microbe-free preparation, MagnaBind Beads can be washed with
antibiotic medium or γ-irradiated. 21354 MagnaBind Goat Anti-Mouse IgG Beads 50 ml

21356 MagnaBind Goat Anti-Rabbit IgG Beads 50 ml Polystyrene Hydrazide Beads
21353 MagnaBind Carboxyl Derivatized Beads 5 ml Ideal for coupling antibodies.
21352 MagnaBind Amine Derivatized Beads 5 ml
Highlights:
21358 MagnaBind Magnet for 96-Well Plate 1 magnet
Separator • This method couples antibodies via carbohydrate groups in the
21357 MagnaBind Magnet for 1.5 ml
Fc region, resulting in site-directed immobilization in one step
1 magnet
Microcentrifuge Tube • Coupling of IgG to Hydrazide Beads can also be done via
21359 MagnaBind Magnet for 6 x 1.5 ml 1 magnet glutaraldehyde activation and reduction of the Schiff base,
Microcentrifuge Tubes upon introduction of an amine-containing species with NaCNBH3
Reference
O’Shannessy, D.J. and Hofmann, W.L. (1987). Biotech. Appl. Biochem. 9, 488–496.

20202 Hydrazide Beads 250 beads
Hydrazide-derivatized, nonporous spherical
polystyrene beads
Bead Diameter: 1/4”
Loading: approximately 3 µmol of hydrazide
function per bead
H
O O
Bead
Bead
NH2 N
N N
H H
Antibody with Carbohydrate

Spacer Hydrazide Groups Oxidized to Form Hydrazone
Arm Group Aldehyde Groups Bond
Hydrazide Bead Structure Oxidized Glycoprotein Immobilized Glycoprotein
Thermo Scientific Affinity Supports
Product Type Product Name Product # Pkg. Size

Antibody Purification – Purify a wide variety of monoclonal and polyclonal antibodies from serum, culture supernatant or ascites fluid.
Protein A Protein A Agarose 20333, 20334 5 ml resin, 25 ml resin
Protein A Columns 20356 5 x 1 ml columns
Protein A IgG Purification Kit 44667 5 x 1 ml columns + reagents
Protein A Trisacryl Resin 20338 5 ml resin
Protein A UltraLink Resin1 53139 5 ml resin
Protein A Plus Agarose 22810, 22811, 22812 5 ml resin, 25 ml resin
Nab Protein A Plus Spin Columns 89952, 89956, 89960 0.2, 1, 5 ml columns
Protein A Plus UltraLink Resin1 53142 5 ml resin
Recombinant Protein A Agarose 20365, 20366 5 ml resin, 25 ml resin
NAb Protein A Spin Kit 89948, 89978 resin + reagents
MagnaBind Protein A Beads 21348 5 ml
Protein G Protein G Agarose 20398, 20399 2 ml resin, 10 ml resin
NAb Protein G Spin Columns 89953, 89957, 89961 2, 1, 5 ml
Protein G UltraLink Resin1 53125, 53126 2 ml resin, 10 ml resin
Protein G UltraLink Columns1 53127 2 x 2 ml columns
Protein G Plus Agarose 22851, 22852 2 ml resin, 10 ml resin
Protein G Plus UltraLink Resin1 53128 2 ml resin
NAb Protein G Spin Kit 89949, 89979 resin + reagents
MagnaBind Protein G Beads 21349 5 ml
Protein A/G Protein A/G Agarose 20421, 20422 3 ml resin, 15 ml resin
NAb Protein A/G Spin Columns 89954, 89958, 89962 2, 1, 5 ml
NAb Protein A/G Spin Kit 89950, 89980 resin + reagents
Protein A/G UltraLink Resin1 53132, 53133 2 ml resin, 10 ml resin
Protein A/G Plus Agarose 20423 2 ml resin
Protein A/G Plus UltraLink Resin 1 53135 2 ml resin
Protein L Protein L Agarose 20510, 20512 2 ml resin
NAb Protein L Spin Columns 89955, 89959, 89963 0.2, 1, 5 ml columns
NAb Protein L Spin Kit 89951, 89981 resin + reagents
Protein L Plus Agarose 20520 2 ml resin
Protein L Chromatography Cartridge 89928, 89929 2 x 1 ml, 1 x 5 ml
Pierce Thiophilic Products Pierce Thiophilic Adsorbent 20500 10 ml resin
Pierce Thiophilic Purification Kit 44916 4 x 3 ml column + reagents
Melon Gel Kits Melon Gel IgG Spin Purification Kit 45206 25 spin columns + reagents
Melon Gel IgG Purification Kit 45212 25 ml resin + reagents
Melon Gel Monoclonal IgG Purification Kit 45214 200 ml resin + reagents
Jacalin Immobilized Jacalin 20395 5 ml resin
MBP Immobilized MBP 22212 10 ml resin
IgM Purification Kit 44897 1 x 5 ml column + reagents
UltraLink Immobilized MBP1 52123 5 ml resin
Protein Isolation Kits – Isolate important subcellular components.

Cell Surface Isolation Cell Surface Protein Isolation Kit 89881 8 spin columns + reagents
Phosphoprotein Enrichment Phosphoprotein Enrichment Kit 90003 10 spin columns + reagents
Ubiquitin Enrichment Ubiquitin Enrichment Kit 89899 15 spin columns + reagents
Glycoprotein Isolation Glycoprotein Isolation Kit, ConA 89804 10 spin columns + reagents
Glycoprotein Isolation Kit, WGA 89805 10 spin columns + reagents
Contaminant Antibody Removal – Minimize background problems by removing cross-reactive antibodies.

Anti-E. coli antibodies Immobilized E. coli Lysate 44938 5 ml resin
Immobilized E. coli Lysate Kit 44940 2 ml column + reagents
Anti-GST antibodies Immobilized GST 20205 2x2 ml column
Immobilized GST 20211 5 ml resin
Immunoprecipitation/Pull-down – Cleanly and efficiently isolate interacting proteins using these innovative products.
Immunoprecipitation Kits Seize Primary IP Kit 45335 Reagents for 20+ IPs
Seize Primary Mammalian IP Kit 45332 Reagents for 20+ IPs
Seize X Protein A IP Kit 45215 Reagents for 40 IPs
Seize X Protein G IP Kit 45210 Reagents for 40 IPs
Pierce Classic Protein A IP Kit 45213 Reagents for 50 IPs
Pierce Classic Protein G IP Kit 45218 Reagents for 50 IPs
Pierce Co-IP Kit 23600 Reagents for 40 IPs
Pierce Mammalian Co-IP Kit 23605 Reagents for 40 IPs
Pull-down Kits Pierce Pull-Down PolyHis Protein:Protein Interaction Kit 21277 Reagents for 25 pull-downs
Pierce Pull-Down GST Protein:Protein Interaction Kit 21516 Reagents for 25 pull-downs
Pierce Pull-Down Biotinylated Protein:Protein Interaction Kit 21115 Reagents for 25 pull-downs
Magnetic Isolation MagnaBind Goat Anti-Mouse IgG Beads 21354 50 ml
MagnaBind Goat Anti-Rabbit IgG Beads 21356 50 ml
MagnaBind Streptavidin Beads 21344 5 ml

Support Approximate Binding Capacity Applications and Features
6% agarose 12–19 mg human IgG/ml resin Purify monoclonal and polyclonal IgG from serum, culture supernatant or ascites fluid
6% agarose 12–19 mg human IgG/ml resin Ideal support to purify rabbit IgG
6% agarose 12–19 mg human IgG/ml resin
Trisacryl® GF-2000 > 15 mg human IgG/ml resin
Polyacrylamide > 16 mg human IgG/ml resin
6% agarose > 35 mg human IgG/ml resin
6% agarose ~1 mg IgG/purification
Iron oxide > 0.2 mg IgG/ml resin Save time by performing immunoprecipitation magnetically
6% agarose 11–15 mg human IgG/ml resin Purify monoclonal and polyclonal IgG from serum, culture supernatant or ascites fluid
6% agarose 11–15 mg human IgG/ml resin Broader species specificity than Protein A with strong binding to Mouse IgG1 and human IgG3
Polyacrylamide > 20 mg human IgG/ml resin Binds only IgG isotype antibodies
6% agarose ~1 mg IgG/purification
Iron oxide > 0.2 mg IgG/ml resin Save time by performing immunoprecipitation magnetically
6% agarose > 7 mg human IgG/ml resin Purify monoclonal and polyclonal IgG from serum, culture supernatant or ascites fluid
6% agarose > 7 mg human IgG/ml resin Broadest specificity-combines the binding specificity of Protein A and Protein G
6% agarose 5–10 mg human IgG/ml resin Purify monoclonal and polyclonal antibodies of all classes from serum, culture supernatant or ascites fluid
6% agarose 5–10 mg human IgG/ml resin Purify single-chain variable fragments (ScFv) or Fab fragments
6% agarose 5–10 mg human IgG/ml resin Binds only to antibodies with specific kappa light chains (mouse k1, human k1, k3, k4)
6% agarose ~20 mg Ig/ml resin Purify antibodies from serum, culture supernatants or ascites fluid of a wide variety of species
6% agarose ~20 mg Ig/ml resin Gentle elution conditions preserve antibody activity
Agarose Purify ~1 mg IgG/spin column Purify IgG from serum in 15 minutes without loss of activity
Agarose Purify ~2 g IgG/kit
Agarose Purify ~1 L culture supernatant Purify IgG from culture supernatant or ascites without loss of activity
6% agarose 1–3 mg human IgA/ml resin Purify human IgA without contaminating IgG or IgM
4% agarose ~1 mg IgM/ml resin Purify IgM in a single step
4% agarose ~1 mg IgM/ml resin
Polyacrylamide > 0.75 mg IgM/ml resin
6% agarose 10–40 million cells/sample Label and purify cell surface proteins from cultured mammalian cells
Agarose ~300 µg phosphoprotein/sample Isolate phosphoproteins from mammalian cells and tissues
Agarose ~0.15 mg protein/sample Isolate poly-ubiquitin modified proteins from cell or tissue lysates
Agarose ~1.5 mg mg protein/sample Isolate glycoproteins with a strong affinity for ConA (mannose and glucose)
Agarose ~1.5 mg mg protein/sample Isolate glycoproteins with a strong affinity for WGA (sialic acid and GlcNAc)
4% agarose ~1 mg E. coli lysate/ml resin Remove E. coli-reactive antibodies to reduce background when screening
4% agarose ~1 mg E. coli lysate/ml resin
6% agarose ~0.5 mg anti-GST/ml resin Prepare antibodies specific to the fusion protein rather than to the fusion tag by removing the GST-reactive antibodies
6% agarose ~0.5 mg anti-GST/ml resin
6% agarose 25–400 µg antibody/IP Isolate proteins using any antibody and without antibody bands interfering with protein analysis on the gel
6% agarose 25–400 µg antibody/IP Enhances co-immunoprecipitation experiments by removing antibody bands from the analysis
6% agarose 50–500 µg antibody/IP Isolate proteins using antibodies that bind to Protein A or G without antibody bands interfering with protein analysis on the gel
6% agarose 50–500 µg antibody/IP Convenient format increases washing efficiency and requires less time than traditional immunoprecipitation
6% agarose 50–500 µg antibody/IP
6% agarose 25–400 µg antibody/IP Control procedures ensure that interactions are real
4% agarose > 10 mg fusion protein/ml resin Isolate interacting proteins more efficiently with a tagged bait protein
4% agarose > 8 mg fusion protein/ml resin Saves time compared to traditional pull-down experiments
4% agarose > 5 mg biotinylated BSA/ml resin
Iron oxide ~0.2 mg mouse IgG/ml resin Save time by performing immunoprecipitation or pull-down experiments magnetically
Iron oxide ~0.2 mg rabbit IgG/ml resin
Iron oxide ~2 µg biotin/ml resin
Thermo Scientific Affinity Supports
Product Type Product Name Product # Pkg. Size

Activated Affinity Supports – Immobilize virtually any molecule on a solid support. Then use it in a batch or column method to purify its binding partners.
Amine-reactive AminoLink Plus Immobilization Kit 44894 5 x 2 ml columns + reagents
AminoLink Plus Coupling Resin 20501 10 ml resin
AminoLink Kit 44890 5 x 2 ml columns + reagents
AminoLink Coupling Resin 20381, 20382 10 ml resin, 50 ml resin
UltraLink Biosupport 53110 2 ml resin
UltraLink Biosupport 53111 10 ml resin
Pierce CDI Support 20259 10 ml resin, 50 ml resin
Pierce CDI Trisacryl (GF-2000) Support 20377 50 ml resin
AminoLink Plus Micro Protein Coupling Kit 20475 10 coupling reactions
Sulfhydryl-reactive SulfoLink Kit for Proteins 44995 5 x 2 ml columns + reagents
SulfoLink Kit for Peptides 44999 5 x 2 ml columns + reagents
SulfoLink Coupling Resin 20401, 20402 10 ml resin, 50 ml resin
UltraLink Iodoacetyl1 53155 10 ml resin
UltraLink Iodoacetyl Micro Peptide Coupling Kit 20485 10 coupling reactions
Carbohydrate-reactive CarboLink Kit 44900 5 x 2 ml columns + reagents
CarboLink Coupling Resin 20391 10 ml resin
UltraLink Hydrazide 53149 10 ml resin
Carboxyl-reactive CarboxyLink Coupling Kit 44899 5 x 2 ml column + reagents
CarboxyLink Coupling Resin 20266 25 ml resin
CarboxyLink Coupling Kit with UltraLink Resin 53154 5 x 2 ml column + reagents
Active hydrogen-reactive PharmaLink Immobilization Kit 44930 5 x 2 ml column + reagents
Antibody Orientation Protein A IgG Plus Orientation Kit 44893 2 x 2 ml column + reagents
Protein G IgG Plus Orientation Kit 44990 2 x 2 ml column + reagents
GST Orientation GST Orientation Kit 78201 2 x 2 ml column + reagents
Fusion Protein Purification – Efficiently purify GST-, PolyHis- or MBP-tagged fusion proteins in a single step.
GST-tagged Immobilized Glutathione 15160 10 ml resin
B-PER GST Fusion Protein Column Purification Kit 78200 5 x 1 ml columns + reagents
B-PER GST Fusion Protein Spin Purification Kit 78400 16 spin columns + reagents
Y-PER GST Fusion Protein Column Purification Kit 78997 5 x 1 ml columns + reagents
PolyHis tagged HisPur Cobalt Resin 89964, 89965, 89966 10 ml, 100 ml, 500 ml resin
HisPur Cobalt Spin Columns 89967, 89968, 89969 25 x 0.2, 5 x 1, 5 x 3 ml columns
Pierce Nickel Chelated Plate2 75824 96-well plate + discs
Pierce Nickel Chelated Discs2 89827 96 discs
B-PER 6xHis Fusion Protein Column Purification Kit 78100 5 x 1 ml columns + reagents
B-PER 6xHis Fusion Protein Spin Purification Kit 78300 16 spin columns + reagents
Y-PER 6xHis Fusion Protein Column Purification Kit 78994 5 x 1 ml columns + reagents
Avidin-Biotin – Purify or immobilize biotinylated molecules through their interaction with avidin.
Avidin Avidin Agarose 20219, 20225 5 ml resin, 25 ml resin
Avidin Columns 20362 5 x 1 ml columns
Streptavidin Streptavidin Agarose 20347, 20349, 20353 2 ml resin, 5 ml resin, 10 ml resin
Streptavidin Columns 20351 5 x 1 ml columns
UltraLink Immobilized Streptavidin1 53113, 53114 2 ml resin, 5 ml resin
UltraLink Immobilized Streptavidin Plus1 53116, 53117 2 ml resin, 5 ml resin
MagnaBind Streptavidin Beads 21344 5 ml
NeutrAvidin Biotin-Binding Protein NeutrAvidin Agarose 29200, 29201 5 ml resin, 10 ml resin
UltraLink Immobilized NeutrAvidin1 53150 5 ml resin
UltraLink Immobilized NeutrAvidin Plus1 53151 5 ml resin
Monomeric Avidin Monomeric Avidin Agarose 20228 5 ml resin
Monomeric Avidin Agarose Kit 20227 2 ml column + reagents
UltraLink Immobilized Monomeric Avidin1 53146 5 ml resin
Biotin Biotin Agarose 20218 5 ml resin
Iminobiotin Agarose 20221 5 ml resin
Specialized Affinity Supports – Purify, or remove from solution, a variety of biologically important molecules.
Phosphorylated Peptides Phosphopeptide Isolation Kit 89853 30 isolations
Albumin Pierce Blue Albumin Removal Kit2 89845, 89846 25 discs, 100 discs + reagents
Heparin-binding Proteins Immobilized Heparin Gel 20207 10 ml resin
C-reactive Protein Immobilized P-Aminophenyl Phosphoryl Choline 20307 5 ml resin
Lectins Immobilized D-Galactose 20372 5 ml resin
Glycoproteins Immobilized Boronic Acid Gel 20244 10 ml resin
His-tagged Proteins Immobilized Iminodiacetic Acid 20277 10 ml resin
Proteases Immobilized Pepstatin 20215 5 ml resin
Immobilized Soybean Trypsin Inhibitor 20235 2 ml resin
Endotoxin DetoxiGel Prepacked Columns 20344 5 x 1 ml columns
DetoxiGel Endotoxin Removing Gel 20339, 20340 10 ml resin, 1,000 ml resin
Detergent Detergent Removing Gel 20208, 20303 10 ml resin, 100 ml resin
All products are available in bulk quantities upon request.
1. UltraLink Support is a copolymer of polyacrylamide and azlactone with high surface area, large pore volume and low nonspecific binding. It is suitable for pressures up to 100 psi and linear flow rates up to 3,000 cm/hour.

Support Approximate Binding Capacity Applications and Features
4% agarose Range of 1–25 mg protein/ml resin Immobilize any protein through exposed lysine residues onto a rigid support for higher flow rates
4% agarose 0.1–2 mg peptide/ml resin Stable, uncharged linkage for maximum binding specificity
4% agarose Range of 1–20 mg protein/ml resin Immobilize any protein through exposed lysine residues
4% agarose 0.1–2 mg peptide/ml resin Stable, uncharged linkage for maximum binding specificity
Polyacrylamide Range of 1–30 mg protein/ml resin Immobilize any protein through exposed lysine residues
Polyacrylamide 0.1–2 mg peptide/ml resin Rigid support for medium pressure applications
6% agarose Range of 1–10 mg protein/ml resin Immobilize any protein through exposed lysine residues
Trisacryl® GF-2000 0.1–2 mg peptide/ml resin Stable, uncharged linkage for maximum binding specificity
4% agarose ~100 µg protein
6% agarose Range of 1–10 mg protein/ml resin Immobilize peptides with a terminal cysteine residue for antibody purification
6% agarose
6% agarose 0.1–2 mg peptide/ml resin Long spacer arm reduces steric hindrance for maximal antibody binding
Polyacrylamide Immobilize proteins that contain free cysteine residues
Polyacrylamide ~250 µg peptide
6% agarose Range of 1–5 mg glycoprotein/ml resin Immobilize polyclonal antibodies and other glycoproteins
6% agarose Antibodies are oriented properly for maximum binding because attachment is through the Fc region
Polyacrylamide
4% agarose Range of 1–10 mg protein/ml resin Immobilize peptides/proteins through aspartic and glutamic acid residues or the carboxy terminus
4% agarose 0.1–2 mg peptide/ml resin Long spacer arm reduces steric hindrance
Polyacrylamide
6% agarose Variable, up to 20 µmol/ml gel Immobilize drugs and other organic molecules with no available reactive groups
6% agarose Up to 16 mg rabbit IgG/column Purify and covalently immobilize an antibody with a single column
6% agarose Up to 16 mg rabbit IgG/column
4% agarose 1–10 mg fusin protein/column Purify and covalently immobilize GST fusion proteins with a single column
4% agarose ~10 mg fusion protein/ml resin Purify GST-tagged fusion proteins

4% agarose ~ 10 mg fusin protein/column Purify GST-tagged fusion proteins
4% agarose ~1 mg fusion protein/spin column Kit includes lysis reagent for optimal protein recovery
4% agarose ~10 mg fusion protein/column
6% agarose ~ 10 mg fusion protein/ml resin Purify His-tagged fusion proteins
6% agarose ~ 10 mg fusion protein/ml resin
Agarose ~1 mg 6xHis-GFP/well
Agarose > 2 mg 6xHis-GFP/disc
4% agarose ~10 mg fusion protein/column Purify His-tagged fusion proteins
4% agarose ~1 mg fusion protein/spin column Kit includes lysis reagent for optimal protein recovery
4% agarose ~10 mg fusion protein/column
6% agarose > 20 µg biotin/ml resin Isolate or immobilize biotinylated proteins, peptides, nucleic acids and other molecules
6% agarose > 20 µg biotin/ml resin
6% agarose 1–3 mg biotinylated BSA/ml resin Isolate or immobilize biotinylated proteins, peptides, nucleic acids and other molecules
6% agarose 1–3 mg biotinylated BSA/column Gives lower background than avidin because it contains no carbohydrate
Polyacrylamide > 2 mg biotinylated BSA/ml resin
Polyacrylamide > 4 mg biotinylated BSA/ml resin
Iron oxide ~2 µg biotin/ml resin Magnetically isolate or immobilize biotinylated proteins, peptides, nucleic acids and other molecules
6% agarose > 20 µg biotin/ml resin Isolate or immobilize biotinylated proteins, peptides, nucleic acids and other molecules
Polyacrylamide 12–20 µg biotin/ml resin Gives lowest background because carbohydrate has been removed and does not contain RYD sequence
Polyacrylamide > 30 µg biotin/ml resin
4% agarose > 1.2 mg biotinylated BSA/ml resin Isolate or immobilize biotinylated proteins, peptides, nucleic acids and other molecules
4% agarose > 1.2 mg biotinylated BSA/ml resin Reversible binding allows mild elution of biotinylated molecules
Polyacrylamide > 1.2 mg biotinylated BSA/ml resin
6% agarose 2 mg avidin/ml resin Isolate or immobilize avidin molecules or conjugates
6% agarose 1 mg avidin/ml resin Reversibly isolate avidin conjugates with mild elution conditions
Agarose 150 µg phosphopeptide/isolation Enrich phosphorylated peptides within a peptide digest for mass spectral analysis
Agarose ~2 mg human serum albumin/disc Remove albumin from antibodies and other samples
4% agarose > 0.2 mg heparin/ml resin Purify a wide variety of proteins that have affinity for heparin
6% agarose > 3 mg human C-reactive protein/ml resin Purify C-reactive protein
6% agarose > 8 mg castor bean lectin/ml resin Purify lectins specific for D-galactose
Polyacrylamide 100 µmol boronate/ml resin Purify glycoproteins, ribonucleosides and other sugar-containing molecules
4% agarose > 14 µmol metal ions/ml resin Purify His-tagged and other metal-binding proteins
6% agarose 1–2 mg pepsin/ml resin Purify pepsin and cathepsins or remove them from a sample
4% agarose Up to 6 mg trypsin/ml resin Purify trypsin, chymotrypsin and elastase or remove them from a sample
6% agarose 2 mg endotoxin Remove endotoxin from protein and nucleic acid samples
6% agarose 2 mg endotoxin/ml resin
Proprietary Varies among detergents – see page 56 Remove detergent from protein and nucleic acid samples
2. SwellGel Support is a convenient, room temperature-stable, dehydrated agarose resin that is rapidly rehydrated when a sample is added. In a 96-well filterplate, SwellGel Resin is ideal for high-throughput purifications.
Appendices
FREE Avidin-Biotin Product Guide

This reference guide brings together
everything needed to biotinylate cell-
surface proteins, purify a biotinylated
target, detect a biotinylated antibody
®
and perform many other applications. It

Thermo Scient fic Pierce
Avidin Biotin Product Guide
includes dozens of references along with

protocols, troubleshooting tips, selection
guides and a complete listing of available
tools. Because the Avidin-Biotin system
can be used in so many ways, you’ll want
to keep this booklet close at hand! To
request a free copy, log on to www.thermo.com/pierce or call
800-874-3723 or 815-968-0747. Outside the United States, contact
your local branch office or distributor.
Antibody Technical Handbook

This 69-page handbook is an essential
resource for any laboratory working with
antibodies. The handbook provides and
overview of antibody structure and types,
Thermo Scient fic Pierce®
Antibody Technical Handbook as well as technical information on the
procedures, reagents and tools used to
produce, purify, fragment and label
antibodies. To request a free copy, log
on to www.thermo.com/pierce or call
800-874-3723 or 815-968-0747. Outside the
United States, contact your local branch
office or distributor.

Thermo Scientific B-PER Technology is protected by U.S. patent #6,174,704.
Thermo Scientific PharmaLink Immobilization Kit Technology is protected by U.S. Patent #5,142,027.
Thermo Scientific Pierce Blue Albumin Removal Technology is protected by U.S. Patent #6,709,743.
Thermo Scientific Pierce Thiophilic Adsorbent Technology is protected by U.S. Patent #4,696,980.
U.S. patent pending on Thermo Scientific Imperial Protein Stain Technology.
U.S. patent pending on Thermo Scientific Melon Gel IgG Spin Purification Kit.
®
Trisacryl is a trademark of Pall Corporation.
™
Luer-Lok is a trademark of Becton, Dickinson and Company.
®
Brij is a registered trademark of ICI Americas.
®
Cibacron is a registered trademark of Ciba Specialty Chemicals, Inc.
®
Triton is a registered trademark of Rohm & Haas Company.
®
Tween is a trademark of ICI Americas.
To order, call 800-874-3723 or 815-968-0747. Outside the United States, contact your local branch office or distributor. 81
Contact Information
Belgium and Europe,

the Middle East
and Africa Distributors
Tel: +32 53 85 71 84
France
Tel: 0 800 50 82 15
The Netherlands
Tel: 076 50 31 880
Germany
Tel: 0228 9125650
United Kingdom
Tel: 0800 252 185
Switzerland
Tel: 0800 56 31 40
Email: perbio.euromarketing@thermofisher.com
www.thermo.com/perbio
United States
Tel: 815-968-0747 or 800-874-3723
Customer Assistance E-mail:
Pierce.CS@thermofisher.com
www.thermo.com
© 2008 Thermo Fisher Scientific Inc. All rights reserved.

1601617 07/08
These products are supplied for laboratory or manufacturing

applications only. Unless indicated otherwise on the inside
back cover, all trademarks are property of Thermo Fisher
Scientific Inc. and its subsidiaries.

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Protein Purification

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®

Thermo Scientiﬁc Pierce

Thermo Scientiﬁc Products for Protein Puriﬁcation

Activated affinity support products and kits enable a researcher to

Affinity purification products using either immobilized ligands

Afﬁnity Puriﬁcation Proteins and other macromolecules of interest can be purified

Antigen 4ml 4ml 4ml

3ml 3ml 3ml

2ml 2ml 2ml

5ml 5ml 5ml

4ml 4ml 4ml

4. Bind anti-antigen 5. Wash off unbound 6. Elute anti-antigen

Typical antibody purification using an immobilized antigen column.

2 For more information, or to download product instructions, visit www.thermo.com/pierce

Common elution systems for protein affinity purification.

Binding and Elution Buffers

Nonspecific (e.g., simple ionic) binding interactions can be

The most widely used elution buffer for affinity purification of

BupH™ Dry Buffers BupH Borate Buffer Packs

The ultimate in integrity Ordering Information

Product # Description Pkg. Size

4 For more information, or to download product instructions, visit www.thermo.com/pierce

Ordering Information Ordering Information

Product # Description Pkg. Size Product # Description Pkg. Size

Ordering Information Product # Description Pkg. Size

BupH Modiﬁed Dulbecco’s PBS Packs

Each pack yields 500 ml of 0.008 M sodium phosphate, 0.002 M

Product # Description Pkg. Size

Porous Beaded Resins

Afﬁnity Puriﬁcation Supports Magnetic Particles

6 For more information, or to download product instructions, visit www.thermo.com/pierce

4% Agarose 6% Agarose Thermo Scientific UltraLink Biosupport

Spin Cups and Columns Spin Columns – Snap Cap

Spin Cups – Paper Filter Ordering Information

69705 Spin Columns – Screw Cap Kit

Disposable Plastic Columns

8 For more information, or to download product instructions, visit www.thermo.com/pierce

Includes accessories plus two each of Product #’s 2ml

• Total Volume: 8 ml (5 ml resin bed, 3 ml reservoir)

centrifuge to efficiently wash away any contaminants and elute 9ml

your purified protein. 6ml

Pierce Centrifuge Columns allow you to use a spin-column format 1ml

Applications for Centrifuge Columns:

Highlights: 89869 Centrifuge Columns, 0.8 ml capacity Kit

69707 Column Extender 10 each

The concept of using immobilized affinity ligands to target

Designing custom affinity supports that are able to target unique

We offer a number of activated affinity supports that are designed

10 For more information, or to download product instructions, visit www.thermo.com/pierce

Capacity Protein Coupling Buffer

12 For more information, or to download product instructions, visit www.thermo.com/pierce

EDC We have developed the Thermo Scientific PharmaLink™ Immobilization

14 For more information, or to download product instructions, visit www.thermo.com/pierce

Thermo Scientific AminoLink Coupling Resin is crosslinked 5 92.7%

Thermo Scientific AminoLink Support detailed immobilization chemistry.

UltraLink Biosupport Pierce CDI Supports

CH2 + H2N Protein N groups per milliliter of resin

UtraLink Biosupport Amine Amide Bond Formation O

Imidazole Amine Carbamate

Product # Description Pkg. Size Ordering Information

20377 Pierce CDI Trisacryl Support 50 ml

16 For more information, or to download product instructions, visit www.thermo.com/pierce

proteins for affinity purification. 75