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Western blotting

Protein gel electrophoresis


technical handbook
separate transfer detect
Prepare samples Choose the electrophoresis
2 Select precast gel and select buffers Select the standard chamber system and power supply Run the gel Stain the gel Post stain 3

Comprehensive solutions Contents


designed to drive your success Electrophoresis overview 4

Select precast gel


Protein gel electrophoresis is a simple way to Gel selection guide 8
separate proteins prior to downstream detection Gels 10

or analysis, and is a critical step in most Prepare samples and select buffers
workflows that isolate, identify, and characterize Sample prep kits 26
Buffers and reagents 28
proteins. We offer a complete array of products Buffers and reagents selection guide 29
to support rapid, reliable protein electrophoresis
Select the standard
for a variety of applications, whether it is the Protein ladders 34
first or last step in your workflow. Our portfolio Protein standards selection guide 36

of high-quality protein electrophoresis products Choose the electrophoresis chamber system and power supply
unites gels, stains, molecular weight markers, Electrophoresis chamber systems 50
Electrophoresis chamber system selection guide 51
running buffers, and blotting products for Power supplies 58
your experiments.
Run the gel
Gel run conditions 59
Troubleshooting tips 60

Stain the gel


Protein stains 62
Protein stains selection guides 63, 67, 69, 70
Electrophoretic staining technology 71

Post stain
Transfer and detection 74

Appendix
Protocol quick reference 76
For a complete listing of all available products
and more, visit thermofisher.com/separate Ordering information 81

Sample preparation and Electrophoresis chamber Electrophoresis


For ordering information refer to page XX. For quick reference on the protocol please refer to page XX. Precast protein gels Protein standards Protein gel stains
electrophoresis buffers systems and power supplies run conditions
Prepare samples Choose the electrophoresis
4 Select precast gel and select buffers Select the standard chamber system and power supply Run the gel Stain the gel Post stain 5

Electrophoresis
Electrophoresis is defined as the Support matrix The acrylamide matrix Electrophoresis conditions
transport of charged molecules Two types of support matrices are commonly used in
Linear vs. gradient gels The separation of molecules is dependent on the electrophoresis
electrophoresispolyacrylamide and agarose. The support conditions. Electrophoresis can be performed under the
Gels that have a single acrylamide percentage are referred to as
through a solvent by an electric matrices act as porous media and behave like a molecular sieve.
linear gels, and those with a range are referred to as gradient
following conditions:
Separation of molecules is dependent upon the gel pore size of
gels. The advantage of using a gradient gel is that it allows the
field. Electrophoresis is a simple, the support matrix used. Agarose has a large pore size and is ideal
separation of a broader range of proteins than a linear gel. Denaturing conditions
for separating macromolecules such as nucleic acids and protein Electrophoresis is performed under denaturing conditions using
rapid, and sensitive analytical complexes. Polyacrylamide has a smaller pore size and is ideal for
Continuous vs. discontinuous gels an anionic detergent such as sodium dodecylsulfate (SDS).
separating most proteins and smaller nucleic acids. SDS denatures and unfolds the protein by wrapping around the
Researchers occasionally refer to gels as continuous or
tool for separating proteins and discontinuous. A continuous gel is a gel that has been formed
hydrophobic portions. SDS binds at a ratio of ~1.4 g SDS per
Polyacrylamide gel electrophoresis from a single acrylamide solution in the entire gel cassette.
gram of protein. The resultant SDS-protein complexes are highly
nucleic acids. Any charged ion or A discontinuous gel is formed from two acrylamide solutions, a
negatively charged and are resolved in the gel based on their size.
(PAGE) small, low-percentage stacking gel where the protein wells reside,
molecule will migrate when placed Polyacrylamide gels are generated by the polymerization of and a larger portion of gel that separates the proteins. In the Nondenaturing (native) conditions
traditional Tris-glycine protein gel system, the proteins are stacked Electrophoresis is performed under nondenaturing (native)
in an electric field. Most biological acrylamide monomers. These monomers are crosslinked into
long chains by the addition of bifunctional compounds such as
in the stacking gel between the highly mobile leading chloride ions conditions using buffer systems that maintain the native protein
(in the gel buffer) and the slower, trailing glycine ions (in the running conformation, subunit interaction, and biological activity. During
molecules carry a net charge at any N,N,-methylenebisacrylamide (bis), which react with the free
functional groups of the chain termini. The concentration of
buffer). The reason for using the stacking gel is to improve the native electrophoresis, proteins are separated based on their
resolution of the bands in the gel. These stacked protein bands charge to mass ratios.
pH other than at their isoelectric acrylamide and bisacrylamide determines the pore size of the gel.
The higher the acrylamide concentration, the smaller the pore size,
undergo sieving once they reach the separating gel.
Reducing conditions
point and will migrate at a rate resulting in resolution of lower molecular weight molecules and
vice versa. Mini vs. midi protein gels Electrophoresis is performed under reducing conditions using
reducing agents such as dithiothreitol (DTT), -mercaptoethanol
proportional to their charge density. PAGE allows one to separate proteins for different applications Commercial gels are available in two size formats, minigels and
midigels. Both gels have similar run lengths, but midigels are wider (-ME) or tris(2-carboxyethyl)phosphine (TCEP).

based on: than minigels, allowing midigels to have more wells or larger wells.
The reducing agents completely unfold the denatured proteins
The mobility of a biological molecule The acrylamide matrix
The additional wells in the midigels permit more samples or large
sample volumes to be loaded onto one gel.
into their subunits by cleaving the disulfide bonds between
cysteine residues.
through an electric field will depend Buffer systems
Buffer systems
on the following factors: Electrophoresis conditions
Electrophoresis is performed using continuous or discontinuous
buffer systems. A continuous buffer system utilizes only one
Field strength buffer in the gel and running buffer. A discontinuous buffer system
utilizes a different gel buffer and running buffer1. This system may
Net charge on the molecule also use two gel layers of different pore sizes and different buffer
composition (the stacking and separating gel). Electrophoresis
Size and shape of the molecule using a discontinuous buffer system results in concentration of the
Did you know?
sample and higher resolution.
Arne Tiselius won the
Ionic strength Nobel Prize in Chemistry
Reference for electrophoretic analysis
Properties of the matrix through which 1. Ornstein L (1964) Disc electrophoresis. 1. Background and theory. Ann N Y Acad Sci 121:321-349. of serum proteins in 1948.

the molecules migrate (e.g., viscosity,


pore size) Mini Gel Tank

Sample preparation and Electrophoresis chamber Electrophoresis


Precast protein gels Protein standards Protein gel stains
electrophoresis buffers systems and power supplies run conditions
Prepare samples Choose the electrophoresis
6 Select precast gel and select buffers Select the standard chamber system and power supply Run the gel Stain the gel Post stain 7

Select precast gel


Comparison of With the Tris-acetate system (Figure 3), three ions are
primarily involved:
High-performance
discontinuous buffer Acetate (), the leading ion from the gel buffer
Tricine (), the trailing ion from the running buffer
precast protein gels
systems
Tris (+), the common ion (in both gel and running buffer)

This system also operates at a significantly lower pH than the Tris-


If you are doing standard one-dimensional protein
SDS-PAGE utilizes a discontinuous buffer system to concentrate glycine system, resulting in less gel-induced protein modifications.
electrophoresis, we have a broad range of solutions to
or stack samples into a very sharp zone in the stacking gel at the
The diagrams below (Figures 1, 2, and 3) summarize the migration fit your research needs. Our selection of precast gels
beginning of the run. In a discontinuous buffer system, the primary
differences in the stacking gel of each system. consists of several different chemistries, well formats,
anion in the gel is different (or discontinuous) from the primary
anion in the running buffer. Both the Invitrogen NuPAGE systems
and gel sizes, so you can get the protein separation
(Bis-Tris and Tris-acetate gels) and the Laemmli (Tris-glycine) you need for accurate downstream results.
Bolt Bis-Tris Plus gel.
system are examples of discontinuous buffer systems and work
in a similar fashion. However, the NuPAGE system operates at a Learn more at thermofisher.com/proteingels
lower pH as a result of the proprietary ions that are in the system. GLYCINE Figure 1. The Tris-glycine gel system.
(Trailing Ion)
Gel buffer ions are Tris and chloride
PROTEIN/SDS COMPLEX
(pH 8.7) Precast gels
In a Tris-glycine system (Figure 1), three ions are primarily involved: (Stacked Proteins)
Running buffer ions are Tris, glycine,
C  hloride (), supplied by the gel buffer, serves as the leading CHLORIDE
(Leading Ion) and SDS (pH 8.3) Popular gel chemistries Specialty gels
ion because it has the highest attraction to the anode relative to PROGRESSION OF RUN
Gel operating pH is 9.5
other anions in the system. Common Ion is Tris, NuPAGE Bis-Tris gels Novex Tricine gels
present in the gel and running buffers
Glycine (), the primary anion provided by the running buffer, NuPAGE Tris-Acetate gels NativePAGE gels
serves as the trailing ion, because it is only partially negatively
charged and remains behind the more highly charged chloride Figure 2. The Bis-Tris gel system.
Bolt Bis-Tris Plus gels Novex IEF gels
MES or MOPS
ions in a charged environment. (Trailing Ion) Gel buffer ions are Bis-Tris and Novex Tris-Glycine gels Novex Zymogram gels
Tris base (+), is a common ion present in both the gel and the PROTEIN/SDS COMPLEX chloride (pH 6.4)
Running buffer ions are Tris, MES or E-PAGE gels
(Stacked Proteins)

running buffers. During electrophoresis, the gel and buffer ions CHLORIDE MOPS, and SDS (pH 7.3) Did you know?
in the Tris-glycine system form an operating pH of 9.5 in the (Leading Ion)
Gel operating pH is 7.0 Over 45 years ago,
separating region of the gel. PROGRESSION OF RUN
Ulrich K. Laemmli first
Common Ion is Bis-tris,
present in the gel
published SDS-PAGE as a
In the case of the Bis-Tris system (Figure 2), three ions are
Casting your own gels? method for cleavage analysis
primarily involved: of structural proteins in
Chloride () supplied by the gel buffer, serves as the fast-moving Figure 3. The Tris-acetate gel system. We offer preassembled empty cassettes, buffers, bacteriophage T4.
TRICINE
leading ion. (Trailing Ion) Gel buffer ions are Tris and acetate and reagents.
(pH 7.0)
MES or MOPS () (depending on the running buffer choice) PROTEIN/SDS COMPLEX
(Stacked Proteins)
Running buffer Ions are Tris, tricine,
serves as the trailing ion. ACETATE and SDS (pH 8.3)
Learn more at
thermofisher.com/gelcastingaccessories
(Leading Ion)
MES: 2-(N-morpholino) ethane sulfonic acid Gel operating pH is 8.1
PROGRESSION OF RUN
MOPS: 3-(N-morpholino) propane sulfonic acid Common Ion is Tris,
Bis-Tris (+) acts as the common ion present in the gel while present in the gel and running buffer

Tris (+) is provided by the running buffer.

The combination of a lower-pH gel buffer (pH 6.4) and running


buffer (pH 7.37.7) leads to a significantly lower operating pH
(pH 7.0) during electrophoresis, resulting in better sample integrity
and gel stability.

Sample preparation and Electrophoresis chamber Electrophoresis


Precast protein gels Protein standards Protein gel stains
electrophoresis buffers systems and power supplies run conditions
Prepare samples Choose the electrophoresis chamber
8 Select precast gel and select buffers Select the standard system and
chamber power
system supply
and power supply Run the gel Stain the gel Post stain Protein gel electrophoresis technical handbook 9

Gel selection guide Find the right gel for your research needs based on molecular weight,
downstream applications, and throughput requirements.
Choose the right gel chemistry for your research needs
Denaturing separation
Bis-Tris chemistry vs. A
1 2 3 4 5 6 7 8 9 10
B
1 2 3 4 5 6 7 8 9 10

Tris-glycine chemistry
Molecular weight

Low molecular weight proteins High molecular weight proteins Broad-range molecular The most widely used gel system for separating
and peptides (>2.5 kDa) (<500 kDa) weight separation a broad range of proteins by SDS-PAGE is
the Laemmli system, which uses Tris-glycine
Novex NuPAGE
Tricine gels Tris-Acetate gels gels comprising a stacking gel component
Figure 4. Protein separation using (A) a Bolt Bis-Tris Plus gel and (B)
that helps focus the proteins into sharp bands
another manufacturers traditional Tris-glycine gel.
Low throughput Medium or high throughput at the beginning of the electrophoretic run and
the resolving gel component that separates the
E-PAGE 48-well proteins based on size. This classic system uses Unlike traditional Tris-glycine gels, NuPAGE and Bolt gels are Bis-
Application or 96-well gels a discontinuous buffer system where the pH and Tris HCIbuffered (pH 6.4) and have an operating pH of about 7.0.
ionic strength of the buffer used for running the gel
Coomassie dye High-sensitivity Downstream applications Large sample volume for (Tris, pH 8.3) is different from the buffers used in the The neutral operating pH of the Bis-Tris systems provides the
or silver staining western blotting requiring high protein integrity high detection sensitivity
(e.g., mass spectrometry)
stacking gel (Tris, pH 6.8) and resolving gel (Tris, following advantages over the Laemmli system:
pH 8.8). The highly alkaline operating pH of the
Longer shelf life of 812 months due to improved gel stability
NuPAGE NuPAGE NuPAGE Bolt Bis-Tris Laemmli system may cause band distortion, loss of
Bis-Tris gels Bis-Tris gels Bis-Tris gels Plus gels resolution, or artifact bands. Improved protein stability during electrophoresis at neutral pH
enabling sharper band resolution and accurate results
Bolt Bis-Tris Bolt Bis-Tris Bolt Bis-Tris
The major causes of poor band resolution with the Laemmli (Moos et al. 1998)
Plus gels Plus gels Plus gels
system are: Complete reduction of disulfides under mild heating conditions
Novex (70C for 10 minutes) and absence of cleavage of Asp-Pro
Tris-Glycine gels Hydrolysis of polyacrylamide at the high pH of the resolving
bonds
gel, resulting in a short shelf life of 8 weeks
Reduced state of the proteins maintained during
Chemical alterations such as deamination and alkylation electrophoresis and blotting of the proteins when using
Native separation
of proteins due to the high pH of the resolving gel Invitrogen NuPAGE Antioxidant
Reoxidation of reduced disulfides from cysteine-containing
proteins, as the redox state of the gel is not constant
Molecular weight Isoelectric point Protease activity
Cleavage of Asp-Pro bonds of proteins when heated at
1st Novex Tris-Glycine Novex Zymogram 100C in Laemmli sample buffer, pH 5.2
dimension NativePAGE gels Novex IEF gels ZOOM IPG strips
gels with native buffers gels (casein,
blue casein, or gelatin

2nd NuPAGE Bis-Tris Novex Tris-Glycine Novex Tris-Glycine Novex Tris-Glycine


substrates)
Choosing the right gel percentage Choosing a well format
dimension gels, 2D well gels, 2D well gels, 2D well ZOOM gels, IPG well
In general, the size of the molecule being separated should and gel thickness
NuPAGE Bis-Tris NuPAGE Bis-Tris dictate the acrylamide or agarose percentage you choose. Use
gels, 2D well ZOOM gels, IPG well a lower percentage gel to resolve larger molecules and a higher We offer most polyacrylamide gels in a choice of nine different well
percentage gel to resolve smaller ones. The exception to this rule formats (17 well, 15 well, 12 well, 10 well, 9 well, 5 well, 1 well,
is when performing isoelectric focusing. Refer to the gel migration 2D/preparative well, or IPG well). Two thicknesses (1.0 mm and
charts throughout this chapter to find the gel best suited for your 1.5 mm) are also available for popular gel types. If loading large
application. As a general rule, molecules should migrate through sample volumes (>30 L), a thicker gel (1.5 mm) with fewer wells
about 70% of the length of the gel for the best resolution. When (e.g., 5 well) or a Bolt gel with its higher-capacity wedge wells
Find the right mini gel using our interactive gel selection tool protein molecular weights are wide ranging, or unknown, gradient is more appropriate. When blotting, remember that proteins will
at thermofisher.com/minigelselection gels are usually the best choice. transfer more easily from a 1.0 mm thick gel than from a 1.5 mm
thick gel.

Sample preparation and Electrophoresis chamber Electrophoresis


Precast protein gels Protein standards Protein gel stains
For ordering information refer to pages 8187. electrophoresis buffers systems and power supplies run conditions
Prepare samples Choose the electrophoresis
10 Select precast gel and select buffers Select the standard chamber system and power supply Run the gel Stain the gel Post stain Protein gel electrophoresis technical handbook 11

Bolt Bis-Tris Plus mini gels Bolt Bis-Tris Plus gels


Figure 7. Bolt Bis-Tris Plus gel migration
Neutral-pH gel system with a unique chart. Optimal separation range is shown within
the gray areas.
wedge well design

Invitrogen Bolt Bis-Tris Plus gels are precast The new Bolt system is wonderful. I am
still amazed that I can run a PAGE gel in
polyacrylamide gels designed for optimal 23 minutes. The entire system is incredibly
separation of a broad molecular weight range of user friendly from the Bolt precast gels with
proteins under denaturing conditions during gel wedged wells for ease of loading to the
Mini Gel Tank system. The bands produced
electrophoresis (Figure 6 and 7). These gels help Specifications from the westerns were sharp and straight.
deliver consistent performance with a neutral-pH Shelf life: ~16 months I would and have highly recommended this
environment to minimize protein degradation. The system to anyone doing protein work.
Average run time: 35 minutes Crystal M., Queens University,
unique wedge well design (Figure 5) allows loading Ontario, Canada
of up to 2x more sample volume than other precast Separation range: 0.3260 kDa
For one of our projects in the lab, we
gels. Bolt gels are ideal for western blot transfer Polyacrylamide concentrations: fixed 8%, 10%, and 12%; resolve proteins by electrophoresis to
and analysis along with any other technique where gradient 412%
determine the accumulation of ubiquitinated
proteins following treatment with a
protein integrity is crucial.
Gel dimensions: 8 x 8 cm (1 mm thick) proteasome inhibitor. When we resolved
the ubiquitinated proteins using the
Bolt Bis-Tris Plus gels offer: Maximum sample volume per 12-well gel: ~40 L, or two-thirds Tris-glycine gels, we observed a smear.
of the sample well volume However, when we switched to resolving
High sample volume capacitywedge well design the ubiquitinated proteins using the Bolt
allows detection of proteins in very dilute samples or Bis-Tris gels, we were delightfully surprised
ts acquired w
sul ith to observe individual protein bands in place
Re
measurement of low-abundance proteins



of the smear. Susan S., University of
Pennsylvania, Philadelphia, US
Preserved protein integrityneutral-pH formulation
minimizes protein modifications
th e k
Mini Gel Tan

Superior band quality and band volume


Invitrogen Novex Bis-Tris Plus chemistry is designed to Figure 6. Bolt Bis-Tris Plus
gel electrophoresis. Protein
deliver sharp, straight bands with higher band volume
standards and samples were
loaded at 10 L sample volumes
Better protein resolutiongels are 10% longer, allowing
in a Bolt 412% Bis-Tris Plus
detection of more protein bands than standard mini gels Gel. Electrophoresis was
performed using the Mini Gel
High lot-to-lot consistencycoefficient of variation (CV) Tank at 200 V (constant). Sharp,
of only 2% for Rf values (migration) straight bands with consistent migration patterns were observed after Did you know?
staining with Invitrogen SimplyBlue SafeStain. Images were acquired Timothy Updyke and
using a flatbed scanner. Lane 1: Invitrogen SeeBlue Plus2 Prestained Sheldon Engelhorn filed a
Standard; Lane 2: 10 g E. coli lysate; Lane 3: Invitrogen Mark12 patent for the neutral-pH Bis-Tris
Unstained Standard (blend of 12 purified proteins); Lane 4: 40 g HeLa cell gel system in 1996.
lysate; Lane 5: 20 g HeLa cell lysate; Lane 6: 5 g BSA; Lane 7:
40 g Jurkat cell lysate; Lane 8: 5 g GST fusion protein; Lane 9:
Invitrogen Novex Sharp Unstained Protein Standard; Lane 10: 5 g Recommended products
Figure 5. The unique wedge well design
-galactosidase. The Invitrogen Bolt Welcome Pack + iBlot 2 System offers a Thermo Scientific Pierce Power
of Bolt Bis-Tris Plus gels. complete protein separation and western blot solution by combining our Stainer is recommended for fast
Mini Gel Tank, Invitrogen Bolt gels and buffers, SeeBlue Plus2 Prestained Coomassie dye staining of
Standard, and Invitrogen iBlot 2 Gel Transfer Device with transfer stacks. Bolt Bis-Tris Plus Gels.

The Bolt Welcome Pack


Learn more at thermofisher.com/bolt + iBlot 2 System.

Sample preparation and Electrophoresis chamber Electrophoresis


For ordering information refer to page 81. For quick reference on the protocol please refer to page 76. Precast protein gels Protein standards Protein gel stains
electrophoresis buffers systems and power supplies run conditions
Prepare samples Choose the electrophoresis
12 Select precast gel and select buffers Select the standard chamber system and power supply Run the gel Stain the gel Post stain Protein gel electrophoresis technical handbook 13

NuPAGE gels NuPAGE gels

A. NuPAGE Bis-Tris gels B. NuPAGE Tris-Acetate gels C. NuPAGE Tris-Acetate gels

Revolutionary high-performance gels (Denaturing separation) (Denaturing separation) (Native separation)

referenced in >20,000 publications

The Invitrogen NuPAGE SDS-PAGE gel system


is a revolutionary high-performance polyacrylamide
gel electrophoresis system that simulates the
denaturing conditions of the traditional Laemmli
system. NuPAGE gels use a unique buffer
formulation to maintain a neutral operating pH Specifications
during electrophoresis, which minimizes protein Shelf life:
modifications that can result in poor band NuPAGE Bis-Tris gels: 16 months
resolution. NuPAGE Tris-Acetate gels: 8 months

Average run time: ~35 minutes


Gels are available in two formulations Invitrogen
NuPAGE Bis-Tris gels are ideal for separating Separation range:
small to midsize proteins while Invitrogen NuPAGE Bis-Tris gels: 1.5300 kDa
NuPAGE Tris-Acetate gels are ideal for separating NuPAGE Tris-Acetate gels: 30400 kDa
large proteins (Figure 8). A gel migration chart is Polyacrylamide concentrations:
shown in Figure 9. NuPAGE Bis-Tris gels: fixed 8%, 10%, and 12%;
gradient 412%
NuPAGE gels are designed for: NuPAGE Tris-Acetate gels: fixed 7%; gradient 38%

Superior protein band resolution and stability Gel dimensions:


neutral-pH environment during electrophoresis minimizes Mini: 8 x 8 cm (1 or 1.5 mm thick)
protein modifications Midi: 8 x 13 cm (1 mm thick)

More efficient western blot transferneutral pH prevents Maximum sample volume per 10-well mini gel: 25 L
reoxidation of reduced samples during protein transfer (1 mm thick); 37 L (1.5 mm thick)

Fast sample run timestypically 3550 minutes

Long product shelf lifestable for 816 months ts acquired w


sul ith
Re

Figure 8. NuPAGE Bis-Tris and Tris-Acetate


gel electrophoresis. Protein standards and
samples were loaded at 10 L sample volumes
A. B.
th e
Mini Gel Tan
k in (A) Invitrogen NuPAGE 412% Bis-Tris and
(B) Invitrogen NuPAGE 38% Tris-Acetate gels. Figure 9. Migration patterns achieved in NuPAGE gels. For optimal gels. (B) Migration patterns of HiMark Unstained Protein Standard on
Electrophoresis was performed using the Mini Gel results, protein bands should migrate within the gray shaded areas. NuPAGE Tris-Acetate gels. (C) Migration pattern for Tris-acetate gel native
Tank at 200 V (constant). Sharp, straight bands were observed after (A) Migration patterns of Invitrogen Novex Sharp Prestained Protein separation is for the Invitrogen NativeMark Unstained Protein Standard.
staining with SimplyBlue SafeStain. Images were acquired using a Standard or Novex Sharp Unstained Protein Standard on NuPAGE Bis-Tris
flatbed scanner. (A and B) Lane 1: SeeBlue Plus2 Prestained Standard;
Lane 2: 10 g E. coli lysate; Lane 3: Mark12 Unstained Standard Recommended products
(blend of 12 purified proteins); Lane 4: 40 g HeLa cell lysate; Lane 5:
Invitrogen HiMark Unstained and Prestained Protein Standards are PageRuler, PageRuler Plus, and Spectra Prestained Protein Ladders
20 g HeLa cell lysate; Lane 6: (A) not used (B) 5 g BSA; Lane 7:
specifically designed for large protein analysis on NuPAGE Tris-Acetate are recommended for use with NuPAGE Bis-Tris gels for easy molecular
40 g Jurkat cell lysate; Lane 8: 5 g GST fusion protein; Lane
gels under denaturing conditions. Both standards offer a ready-to-load weight determination.
9: Novex Sharp Unstained Protein Standard; Lane 10: 5 g
-galactosidase. format and consist of 9 proteins with a size range of 40500 kDa.
Visualize with Coomassie stain, silver stain, or fluorescent protein
Learn more at thermofisher.com/nupage  stains after electrophoresis (see Stain the gel, page 62).

Sample preparation and Electrophoresis chamber Electrophoresis


For ordering information refer to page 81. For quick reference on the protocol please refer to page 76-77. Precast protein gels Protein standards Protein gel stains
electrophoresis buffers systems and power supplies run conditions
Prepare samples Choose the electrophoresis
14 Select precast gel and select buffers Select the standard chamber system and power supply Run the gel Stain the gel Post stain Protein gel electrophoresis technical handbook 15

Novex Tris-Glycine gels Novex Tris-Glycine gels

A. Denaturing separation B. Native separation


Laemmli-based precast gels for high Tris-Glycine Gels

efficiency, reproducibility, and performance Large proteins*


(116500 kDa)
Mid-size proteins
(20250 kDa)
Small proteins
(360 kDa)
Wide range
(6200 kDa)
4% 6% 8% 10% 12% 14% 16% 18% 412% 816% 420% 1020%
0%
500 kDa

Invitrogen Novex Tris-Glycine gels are based on Specifications 260


260kDa
kDa
500 kDa
traditional Laemmli protein electrophoresis with Shelf life: 12 months 10%
minor modifications for maximum performance in 260 kDa 160 kDa

the precast format. These gels provide reproducible Run time: ~90 minutes 260 kDa 260 kDa
260 kDa
290 kDa 160 kDa 260 kDa
separation of a wide range of proteins into well- 20% 260 kDa 110 kDa
Separation range: 6500 kDa 260 kDa
160 kDa 160 kDa 110 kDa
260 kDa
resolved bands (Figure 10). A gel migration chart is Polyacrylamide concentrations:
160 kDa 110 kDa
110 kDa 80 kDa 80 kDa
80 kDa 60 kDa
shown in Figure 11. Fixed concentrations available from 4% to 18% 30%
240 kDa
160 kDa 110 kDa
80 kDa
60 kDa
50 kDa
160 kDa
40 kDa 60 kDa
290 kDa
Gradient gels with ranges of 412%, 420%, 816%, 160 kDa
60 kDa
50 kDa 260 kDa 110 kDa
80 kDa
Novex Tris-Glycine gels are: and 1020%
110 kDa
40 kDa 50 kDa
50 kDa 160 kDa
40% 30 kDa 80 kDa
Individually packaged for convenience Gel dimensions: 240 kDa
60 kDa
40 kDa
80 kDa 40 kDa
60 kDa
Mini: 8 x 8 cm (1 or 1.5 mm thick) 110 kDa 160 kDa
Compatible with most protein standards for accurate size 160 kDa
50 kDa
30 kDa
110 kDa
Midi: 8 x 13 cm (1 mm thick) 50% 50 kDa
determination 20 kDa
80 kDa
60 kDa 110 kDa
Maximum sample volume per well: 25 L (1 mm thick); 40 kDa
40 kDa
Flexible for use with native or denatured protein samples, 80 kDa 60 kDa 30 kDa
37 L (1.5 mm thick) 60% 30 kDa 15 kDa
with specially formulated buffers for each condition 116 kDa
20 kDa
30 kDa
110 kDa
50 kDa 50 kDa
10 kDa
40 kDa 20 kDa
60 kDa 15 kDa
ts acquired w 70% 20 kDa
sul ith 30 kDa 60 kDa
Re 160 kDa
20 kDa
97 kDa



40 kDa
15 kDa
10 kDa 50 kDa
15 kDa
40 kDa 30 kDa 10 kDa
80% 15 kDa
50 kDa
10 kDa
th e k
Mini Gel Tan 20 kDa 20 kDa
10 kDa 15 kDa
166 kDa 66 kDa
90% 30 kDa 30 kDa 10 kDa
15 kDa
Figure 10. Novex Tris-Glycine 40 kDa
20 kDa
gel electrophoresis. Protein 55 kDa
15 kDa
97 kDa 10 kDa
standards and samples were 100%
loaded at 10 L sample volumes
in 420% Tris-Glycine gels. Figure 11. Migration patterns of protein molecular weight standards in Novex Tris-glycine gels. For optimal results, protein bands should
Electrophoresis was performed migrate within the gray shaded areas. (A) *Migration patterns of HiMark Unstained Protein Standard. Migration patterns of Novex Sharp Pre-
using the Mini Gel Tank at Stained Protein Standard or Novex Sharp Unstained Protein Standard. (B) Migration pattern of NativeMARK Unstained Protein Standard.
200 V (constant). Sharp, straight bands were observed after staining with
SimplyBlue SafeStain. Images were acquired using a flatbed scanner. Lane
1: SeeBlue Plus2 Prestained Standard; Lane 2: 10 g E. coli lysate; Lane 3:
Mark12 Unstained Standard (blend of 12 purified proteins); Lane 4: 40 g
HeLa cell lysate; Lane 5: 20 g HeLa cell lysate; Lane 6: 5 g BSA; Lane
7: 40 g Jurkat cell lysate; Lane 8: 5 g GST fusion protein; Lane 9: Novex Recommended products
Sharp Unstained Protein Standard; Lane 10: 5 g -galactosidase.
 or sample cleanup prior to electrophoresis, we recommend using the
F Buffers for native proteins: Invitrogen Novex Tris-Glycine Native
Pierce SDS-PAGE Sample Prep Kit. Sample Buffer and Novex Tris-Glycine Native Running Buffer.

 uffers for denatured proteins: Invitrogen Novex Tris-Glycine SDS


B PageRuler, PageRuler Plus, and Spectra protein ladders
Sample Buffer and Novex Tris-Glycine SDS Running Buffer. are recommended for molecular weight determination with
Novex Tris-Glycine gels.


Learn more at thermofisher.com/trisglycine

Sample preparation and Electrophoresis chamber Electrophoresis


For ordering information refer to page 8283. For quick reference on the protocol please refer to page 77-78. Precast protein gels Protein standards Protein gel stains
electrophoresis buffers systems and power supplies run conditions
Prepare samples Choose the electrophoresis
16 Select precast gel and select buffers Select the standard chamber system and power supply Run the gel Stain the gel Post stain Protein gel electrophoresis technical handbook 17

NativePAGE gels NativePAGE gel

Superior resolution of native proteins and


protein complexes

The Invitrogen NativePAGE Bis-Tris gel system


is based on the blue native polyacrylamide gel Specifications
electrophoresis (BN PAGE) technique that uses
Shelf life: 6 months
Coomassie G-250 dye as a charge shift molecule
that binds to proteins and confers a negative Average run time: 90 minutes Did you know?
charge without denaturing the proteins (Figure Separation range: 1510,000 kDa The blue native polyacrylamide
12). This technique overcomes the limitations gel electrophoresis technique
Polyacrylamide concentrations: gradient 312% and 416% was developed by Hermann
of traditional native electrophoresis by providing
Schagger and Gebhard von
a near-neutral operating pH and detergent Gel dimensions: 8 x 8 cm (1 mm thick)
Jagow in 1991.
compatibility. The near-neutral (pH 7.5) environment Maximum sample volume per 10-well gel: 25 L
of the NativePAGE system during electrophoresis
Figure 13. NativePAGE gel migration chart.
results in maximum protein and gel matrix stability, Migration patterns of the NativeMark Unstained
enabling better band resolution than other native Protein Standard on NativePAGE gels
Figure 12. NativePAGE gel
gel systems. A gel migration chart is shown in electrophoresis. Two-fold
are shown.
Figure 13. Is there a higher res pic dilution series of protein extracts
somewhere? I copied and were run on an Invitrogen
pasted this from source file. NativePAGE Novex 312% Bis-
The NativePAGE gel system is designed for: Tris Protein Gel using a Mini Gel
Tank. Following electrophoresis,
A wide resolving rangefrom 15 kDa to over 10 MDa
the gel was stained with
(Figure 12), regardless of isoelectric point Coomassie dye and imaged
using a flatbed scanner. Lanes 1
Neutral-pH separationthe native state of protein com- and 10: blank; Lanes 2 and 6:
plexes is better preserved 5 L NativeMark Unstained
ts acquired w
sul ith
Re Protein Standard; Lanes 3, 4

Superior performancehigher resolution than Tris-glycine and 5: 10, 5, and 2.5 g spinach
native electrophoresis chloroplast extract; Lanes 7, 8
and 9: 10, 5, and 2.5 g bovine

th e
Mini Gel Tan
k mitochondrial extract.
 dvantages of the NativePAGE gel system over
A
the Tris-glycine gel system include:
Reduced vertical streakingCoomassie G-250 dye binds
to nonionic detergent molecules in the sample and carries
them in the dye front, ahead of resolving proteins

Better separation of proteinspositively charged pro-


Recommended products
teins with high isoelectric points are converted to proteins
with a net negative charge, allowing migration to the anode NativeMark Unstained Protein Standard is recommended for use
with native gel chemistries, including our Tris-glycine, Tris-acetate, and
Minimized protein aggregationCoomassie G-250 dye NativePAGE gel systems. This standard offers a wide molecular weight
binding allows separation of membrane proteins and range of 201,200 kDa, and the 242 kDa -phycoerythrin band is visible as
a red band after electrophoresis for reference (prior to staining). See page
proteins with exposed hydrophobic areas
40 for details.

Learn more at thermofisher.com/nativepage

Sample preparation and Electrophoresis chamber Electrophoresis


For ordering information refer to page 84. For quick reference on the protocol please refer to page 78. Precast protein gels Protein standards Protein gel stains
electrophoresis buffers systems and power supplies run conditions
Prepare samples Choose the electrophoresis
18 Select precast gel and select buffers Select the standard chamber system and power supply Run the gel Stain the gel Post stain Protein gel electrophoresis technical handbook 19

Novex Tricine gels Novex Tricine gel

High-resolution gels for peptide analysis


and low molecular weight proteins

The Invitrogen Novex Tricine gel system is a Specifications


modification of the Tris-glycine system in which Shelf life: 12 months
tricine replaces glycine in the running buffer.
Average run time: 90 minutes
This system uses a discontinuous buffer system
specifically designed for the resolution of low Separation range: 220 kDa Did you know?
molecular weight proteins (Figure 14). Sample preparation is not
Polyacrylamide concentrations: fixed 10% and 16%;
the only factor that can result
gradient 1020% in poorly resolved bands.
Advantages of Novex Tricine gels over
Gel dimensions: 8 x 8 cm (1 mm thick) You can minimize protein
Tris-glycine gels include: degradation by using gels
Maximum sample volume per 10-well gel: 25 L with neutral-pH chemistry.
Increased resolution of proteins with molecular weights
Figure 15. Novex Tricine gel migration chart.
as low as 2 kDa (Figure 15)
For optimal resolution, protein bands should
ts acquired w
sul migrate within the shaded areas.
Improved compatibility with direct protein sequencing Re
ith



applications after transferring to PVDF membranes

Minimized protein modification due to the lower pH of


th e k
the tricine buffering system Mini Gel Tan

Figure 14. Novex Tricine


Good to know
gel electrophoresis. Protein
standards and samples
How Novex Tricine gels work were loaded at 10 L sample
volumes on Invitrogen Novex
1020% Tricine Protein Gels.
In the traditional Tris-glycine protein gel system, the resolution Electrophoresis was performed
of smaller proteins (<10 kDa) is hindered by the continuous using the Mini Gel Tank at 200 V (constant). Sharp, straight bands were
accumulation of free dodecyl sulfate (DS) ions from the SDS observed after staining with SimplyBlue SafeStain. Images were acquired
using a flatbed scanner. Lane 1: SeeBlue Plus2 Prestained Standard; Lane
sample and running buffers in the stacking gel, which causes 2: 10 g E. coli lysate; Lane 3: Mark12 Unstained Standard (blend of 12
mixing of the DS ions with smaller proteins and results in fuzzy purified proteins); Lane 4: 40 g HeLa cell lysate; Lane 5: 20 g HeLa cell
bands and decreased resolution. The mixing also interferes lysate; Lane 6: 5 g BSA; Lane 7: 40 g Jurkat cell lysate; Lane 8: 5 g
GST fusion protein; Lane 9: Novex Sharp Unstained Protein Standard; Lane
with the fixing and staining of smaller proteins. The Novex 10: 5 g -galactosidase.
Tricine gel system uses a low pH in the gel buffer and sub-
stitutes tricine for glycine in the running buffer. The smaller
proteins and peptides that migrate with the stacked DS ions Recommended products
in the Tris-glycine gel system are well separated from DS ions Use Novex Tricine gels with our In-Gel Tryptic Digestion Kit for separation
in the Novex Tricine gel system, offering sharper bands and and digestion of peptides for mass spectrometry.
higher resolution.

Learn more at thermofisher.com/tricine

Sample preparation and Electrophoresis chamber Electrophoresis


For ordering information refer to page 85. For quick reference on the protocol please refer to page 79. Precast protein gels Protein standards Protein gel stains
electrophoresis buffers systems and power supplies run conditions
Prepare samples Choose the electrophoresis
20 Select precast gel and select buffers Select the standard chamber system and power supply Run the gel Stain the gel Post stain Protein gel electrophoresis technical handbook 21

Novex IEF gels Novex IEF gel

Precast gels for isoelectric Separated on


precast vertical
point determination gel (slab)
Cathode

Isoelectric focusing (IEF) is an electrophoresis Specifications


technique that separates proteins based on their Shelf life: 2 months
isoelectric point (pI). The pI is the pH at which a Did you know?
Average run time: 2.5 hours Harry Svensson-Rilbe and
protein has no net charge and does not move in an
his student Olof Vesterberg
electric field. Invitrogen Novex IEF gels effectively Separation range: first described the theory
create a pH gradient so proteins separate pH 310 gels: pI performance range is 3.58 of separation of amphoteric
according to their unique pI (Figure 16 and 17). pH 37 gels: pI performance range is 3.07.0 proteins along a pH gradient
These gels can be used for pI determination or by applying an electric field
Polyacrylamide concentration: fixed 5%
for detection of minor changes in a protein due to in the 1960s.
Gel dimensions: 8 x 8 cm (1 mm thick)
deamination, phosphorylation, or glycosylation, and
can resolve different proteins of similar size that Maximum sample volume per 10-well gel: 20 L
cannot be resolved on standard SDS-PAGE gels.
ts acquired w
sul ith
Re
When used with our convenient, pre-optimized



Anode +
1 2 3 4 5 6 7 8 9 10
buffers, solubilizers, and molecular weight pl

markers, Novex IEF gels can provide: 8.0 Figure 17. Novex IEF gel migration chart using

th e
nk
the Novex IEF marker. Proteins shown are
Mini Gel Ta
Accurate pI determination 1: amyloglucosidase (Aspergillus niger), pI = 3.5;
2: glucose oxidase (Aspergillus niger), pI = 4.2;
Clear, sharp bands for easy identification of 7.4 3: trypsin inhibitor (soybean), pI = 4.5; 4a and 4c:
protein modifications Figure 16. Novex IEF -lactoglobulin (bovine, milk), pI = 5.2 and 5.3;
gel electrophoresis. 5: carbonic anhydrase (bovine, erythrocytes), pI
Higher resolution of slight differences in size when used in 6.0
A 2-fold dilution series = 6.0; 6a and 6c: myoglobin (horse, muscle), pI =
of IEF Marker 310 6.9 and 7.4; 7a, 7m and 7c: lectin (Lens culinaris),
combination with SDS-PAGE for 2D electrophoresis
was run in duplicate pI = 7.8, 8.0 and 8.3; 8: ribonuclease A (bovine,
4.5 on an Invitrogen pancreas), pI = 9.5; and 9: cytochrome c (horse,
Novex pH 310 IEF heart), pI = 10.7.
Protein Gel using
a Mini Gel Tank. The IEF Marker 310 consists of proteins with a variety
of isoelectric points; these proteins include lectin (pI = 7.8, 8.0, and 8.3),
myoglobin from horse muscle (pI = 6.9 and 7.4), carbonic anhydrase from
bovine erythrocytes (pI = 6.0), -lactoglobulin from bovine milk (pI = 5.2 and
5.3), soybean trypsin inhibitor (pI = 4.5), and glucose oxidase (pI = 4.2). After
electrophoresis, the gel was fixed and stained using Coomassie R-250 dye.
Gel imaging was performed with a flatbed scanner. Volume of IEF Marker
310 loaded: Lanes 1 and 6: 20 L; Lanes 2 and 7: 10 L; Lanes 3 and
8: 5 L; Lanes 4 and 9: 2.5 L; Lanes 5 and 10: blank.

Recommended products
Novex IEF buffer kitsincludes optimized cathode, anode, and sample ZOOM IEF Fractionator Combo Kit
buffers to reduce variability and enable consistent results. offers a fast, reliable method to reduce
sample complexity, enrich low-abundance
IEF Marker 310ready to use, enables accurate results. proteins, and increase the dynamic range
of detection.

Learn more at thermofisher.com/ief

Sample preparation and Electrophoresis chamber Electrophoresis


For ordering information refer to page 85. For quick reference on the protocol please refer to page 79. Precast protein gels Protein standards Protein gel stains
electrophoresis buffers systems and power supplies run conditions
Prepare samples Choose the electrophoresis
22 Select precast gel and select buffers Select the standard chamber system and power supply Run the gel Stain the gel Post stain Protein gel electrophoresis technical handbook 23

Novex Zymogram gels Table 1. Novex Zymogram gels available.


Novex Zymogram gel
Novex
Novex Novex
Zymogram
Easy in-gel protease analysis Zymogram Zymogram
blue casein Protease analysis
gelatin gel casein gel
gel 416% gel
10% gel 12% gel
Invitrogen Novex Zymogram gels are excellent (w/gelatin) (w/casein)
(w/prestained
casein blue)
Gel 10% Tris- 12% Tris- 416% Tris-
tools for detecting and characterizing proteases composition Glycine gel Glycine gel Glycine gel
that utilize casein or gelatin as a substrate.
Casein and gelatin are the most commonly Substrate 0.1% gelatin 0.05% casein 0.1% casein, 10
used substrates for demonstrating the activity with blue stain
incorporated
of proteases. Novex Zymogram gels are used
in gel
to analyze a variety of enzymes, including matrix 20
metalloproteinases, lipases, and other proteases
(Figure 18). Available gel types are shown in Table 1. Sensitivity 106 units of 7 x 10 4 units 1.5 x 10 3 units
collagenase of trypsin of trypsin
30
Good to know
1
Post-staining Yes Yes No
How do Novex Zymogram gels work?
required? 40

Protease samples are denatured in SDS buffer under nonre- Separation 20120 kDa 30150 kDa 10220 kDa
ducing conditions and without heating, and run on a Novex

% of length of gel
range
50
Zymogram gel using Novex Tris-Glycine SDS Running Buffer.
2 1 Figure 19. Novex Zymogram gel
After electrophoresis, the enzyme is renatured by incubating
migration chart. The numbered bands
the gel in Invitrogen Novex Zymogram Renaturing Buffer Specifications refer to the following proteases:
60
2 2 Band 1: Collagenase Type I (140 kDa)
that contains a nonionic detergent. The gels are then equili- Shelf life: 2 months
Band 2: Thermolysin (37 kDa)
brated in Invitrogen Novex Zymogram Developing Buffer
Average run time: 90 minutes 3 3 Band 3: Chymotrypsin (30 kDa)
to add divalent metal cations required for enzymatic activity, Band 4: Trypsin (19 kDa)
70
and then stained and destained. Regions of protease activity Separation range: 10220 kDa (Figure 19) 4
3
appear as clear bands against a dark blue background where Polyacrylamide concentrations: fixed 10% (with gelatin), fixed 4
the protease has digested the substrate. 12% (with casein); gradient 416% (with blue casein) 80 2

Gel dimensions: 8 x 8 cm (1 mm thick)

Maximum sample volume per well: 20 L 4


90

ts acquired w
sul ith
Re

1 2 3 4 5 6 7 8 9 10 Figure 18. Novex Zymogram gel electrophoresis. 4


100
Type I collagenase was run in duplicate on an
Invitrogen Novex 10% Zymogram (Gelatin) Protein

th e
Mini Gel Tan
k Gel using a Mini Gel Tank. The gel was developed
using Novex Zymogram Renaturing Buffer and Novex
Zymogram Developing Buffer and stained using SimplyBlue
SafeStain. Images were acquired using a flatbed scanner. Lanes 3 and 7:
Recommended products
5 L of 2.0 U/mL type I collagenase; Lanes 1, 4, 5, and 10: 12 L SeeBlue After electrophoresis, incubate the gel in Zymogram Renaturing
Prestained Protein Standard. Buffer to renature the enzyme. The gels are then equilibrated in
Zymogram Developing Buffer to add divalent metal cations required
for enzymatic activity.

Learn more at thermofisher.com/zymogram

Sample preparation and Electrophoresis chamber Electrophoresis


For ordering information refer to page 85. For quick reference on the protocol please refer to page 80. Precast protein gels Protein standards Protein gel stains
electrophoresis buffers systems and power supplies run conditions
Prepare samples Choose the electrophoresis
24 Select precast gel and select buffers Select the standard chamber system and power supply Run the gel Stain the gel Post stain Protein gel electrophoresis technical handbook 25

E-PAGE High-Throughput E-PAGE gel

Precast Gel System


E-PAGE E-PAGE
48 8% Gel 96 6% Gel
Protein separation and analysis for 0% 0%

increased sample throughput


220 kDa
10%
25%

The Invitrogen E-PAGE High-Throughput



220 kDa 120 kDa
Did you know?
20% Our E-Base devices are
Precast Gel System is designed for fast, bufferless 60 kDa
120 kDa 50% compatible with the Society for
medium- and high-throughput protein analysis. 100 kDa
40 kDa
Biomolecules Screening (SBS)
30%
Invitrogen E-PAGE 48-well and 96-well precast standard plate size and can be
gels consist of a buffered gel matrix and electrodes 80 kDa
75% conveniently mounted on liquid
20 kDa Figure 21. E-PAGE gel migration chart.
packaged inside a disposable, UV-transparent 40% handling robot decks.
Specifications 60 kDa
Migration patterns of the Invitrogen E-PAGE
cassette. Each cassette is labeled with a unique 50 kDa
MagicMark Unstained Protein Standard
barcode to facilitate identification of the gel using Shelf life: 6 months 50% 100% are shown.

commercial barcode readers. These gels can be Average run time: 14 minutes 40 kDa

loaded by multichannel pipettor or automated Separation range: 10200 kDa


60%

loading system. The E-PAGE system also includes 30 kDa

E-Base integrated devices to run the gels, an Polyacrylamide concentrations: 70%


E-PAGE 48 gel: fixed 8%
E-Holder platform for optional robotic loading, and 20 kDa
E-PAGE 96 gel: fixed 6%
free E-Editor 2.0 Software to align images for easy 80%
comparison. Gel dimensions: 13.5 x 10.8 cm (3.7 mm thick)

Maximum sample volume per well: 90%


Advantages of using the E-PAGE High-Throughput E-PAGE 48 gel: 20 L
Precast Gel System include: E-PAGE 96 gel: 15 L 100%

Ease-of-usequick setup and fast protein separation in


A B
about 23 minutes

Fast loadingcompatible with multichannel pipettors and


robotic loading
Recommended products
Efficient western blotting and stainingoptimized The E-PAGE SeeBlue Prestained Protein Standard or E-PAGE
protocols and reagents MagicMark Unstained Protein Standard are specifically designed for use
with E-PAGE gels.

Good to know C

Mother E-Base E-PAGE 96 gels


How do E-PAGE gels work?

E-PAGE gels run in the Invitrogen E-Base electrophoresis de- Daughter E-Base

vice, which has an integrated power supply for direct connec-


tion to an electrical outlet. Use the Invitrogen Mother E-Base
Figure 20. Loading and running E-PAGE gels. (A) Loading E-PAGE 48
device for a single E-PAGE gel, or use the Mother E-Base gels using a multi-channel pipettor. (B) Loading E-PAGE 96 gels using a
device in conjunction with two or more Invitrogen Daughter multi-channel pipettor. (C) The Mother/Daughter E-Base combination.
E-Base devices for running multiple gels simultaneously.

Learn more at thermofisher.com/epage

Sample preparation and Electrophoresis chamber Electrophoresis


For ordering information refer to page 85. Precast protein gels Protein standards Protein gel stains
electrophoresis buffers systems and power supplies run conditions
Prepare samples Choose the electrophoresis
26 Select precast gel and select buffers Select the standard chamber system and power supply Run the gel Stain the gel Post stain Protein gel electrophoresis technical handbook 27

Prepare the sample


Pierce SDS-PAGE Sample treated with the Pierce

Sample prep kits


Untreated SDS-PAGE Sample Prep Kit
M S M S M S M S
High salt concentration in samples: High salt concentrations
result in increased conductivity that affects protein migration, and Sample Prep Kit
Before a sample can be loaded can result in gel artifacts in adjacent lanes containing samples with
normal salt concentrations. Perform dialysis or precipitate and Quick protein clean-up and enrichment
onto a gel for analysis, it must resuspend samples in lower-salt buffer prior to electrophoresis.
for SDS-PAGE
be properly prepared. Depending Guanidine-HCl in samples: Samples solubilized in guanidine-
HCl have high ionic strength, and produce increased conductivity A protein sample can be purged of any contaminants
on the gel type, this may involve similar to high salt concentrations. In addition, guanidine typically in only 10 minutes using the Thermo Scientific
Figure 22. Minimize distortion caused by detergents. Rat C6 cells were
precipitates in the presence of SDS leading to various types of gel Pierce SDS-PAGE Sample Prep Kit. This is much lysed and a membrane protein fraction isolated using the Thermo Scientific
denaturing the proteins, reducing artifacts. If possible, change the solubilization agent by dialysis faster than dialysis or ultrafiltration and yields higher MemPER Eukaryotic Membrane Protein Extraction Reagent. Membrane
and hydrophilic cell fractions were separated by SDS-PAGE using 420%
prior to electrophoresis. protein recoveries while concentrating the sample.
any disulfide bonds, adjusting gradient gels with or without prior treatment using the Pierce SDS-PAGE
Sample Prep Kit. Western blot analysis was performed using an antibody

the ionic strength, and removing Cell lysates Advantages of using the Pierce SDS-PAGE Sample against cytochrome oxidase subunit 4 (COX4) and Thermo Scientific
SuperSignal West Femto chemiluminescent substrate. Kit-treated samples
Prep Kit include:
exhibit better band straightness and resolution with low molecular weight
interfering contaminants. General Consider the following when performing electrophoresis of
cell lysates:
Eliminates artifacts caused by incompatible proteins than samples that were untreated.
S = Soluble fraction (hydrophilic) M = Membrane fraction
contaminantsremoves dyes, reducing agents, deter-
guidelines for preparing samples Genomic DNA in the cell lysate may cause the sample to
gents, sugars, glycerol, guanidine, urea, and ammonium 100 Figure 23.
sulfate to provide reproducible results for SDS-PAGE Consistent
become viscous and affect protein migration patterns and
are provided below.

Percent protein recovered


80 88% protein
analysis (Figure 22) 85%
resolution. Shear genomic DNA to reduce viscosity before 75% 77% 77% recovery is
74%
loading the sample. Compatible with the Thermo Scientific Pierce BCA 60 achieved
Assayallows quantification of the processed sample using the
General guidelines for Cells lysates contain soluble and insoluble fractions. The size of
each fraction depends on the type of sample being analyzed.
40 Pierce SDS-
PAGE Sample
Enriches dilute protein solutionsconcentrates protein
preparing samples: The nature of the insoluble fraction may result in altered sample by eight-fold in less than 20 minutes for SDS-PAGE 20 Prep Kit.
Pure proteins
protein migration patterns and resolution. Separate the two analysis (Figure 22)
0 (60 g) of
fractions by centrifugation and load them on separate lanes for Carbonic Ovalbumin Transferrin Ubiquitin Cytochrome c Bacterial assorted
Prepare your sample in the appropriate sample buffer such that Fast and easy to use for up to 70 g of protein per
electrophoresis. anhydrase lysat e molecular mass
the final concentration of the sample buffer is 1X. Recommended sampleuses new spin cup format that allows higher (30, 44, 80, 86, and 120 kDa) as well as a bacterial lysate were processed
sample buffers are listed on page 29. If radioimmunoprecipitation assay (RIPA) buffer is used in cell amounts of protein to be processed than with the original using this kit. Protein concentrations were determined with the Pierce BCA
lysis, subsequent blotting of proteins less than 40 kDa may be procedure Protein Assay Kit and reported as percent protein recovered.
inhibited due to the presence of Triton X-100 in the buffer.
Running reduced and non-reduced samples: For optimal Table 2. Interfering substances effectively removed.
results, we do not recommend running reduced and non-reduced For quick protein clean-up and enrichment for SDS-PAGE we Good does
How to know
it work?
samples on the same gel. If you do choose to run reduced and recommend using the Thermo Scientific Pierce SDS-PAGE Sample Percent protein recovered
Interfering reagents (Starting amount = 20 g BSA)
non-reduced samples on the same gel, do not run reduced and Prep Kit, which removes substances such as guanidine-HCL Our Pierce SDS-PAGE Sample Prep Kit uses a unique resin
Control (water) 75%
non-reduced samples in adjacent lanes. The reducing agent may and ionic detergents that can result in protein bands that appear of modified diatomaceous earth that binds protein in DMSO. 0.5 M Sodium chloride 80%
have a carry-over effect on the non-reduced samples if they are in smeared or wavy in the gel or on a western blot. Simply combine 2300 L of sample containing up to 70 g of 2 M Ammonium sulfate 76%
close proximity. protein with 20 L of Pierce SDS Protein Binding Resin and 20% SDS 75%
DMSO. After the proteins bind to the resin, wash away the 10% Triton detergent 75%

Heating samples: Heating the sample at 100C in SDS-containing unbound contaminating chemicals. Finally, elute the sample 6 M Urea:DMSO (1:3 ratio) 75%
1 M Sodium chloride 75%
buffer results in proteolysis (Kubo, 1995). We recommend heating in 50 L of the Elution Buffer. The recovered protein sample is
6 M Urea 74%
samples for denaturing electrophoresis (reduced or non-reduced) ready to mix with the supplied Sample Loading Buffer for
10% CHAPS 80%
at 85C for 25 minutes for optimal results. Do not heat the gel loading. 25% Glycerol 71%
samples for non-denaturing (native) electrophoresis or Novex 10% OTG 71%
Zymogram Gels. Learn more at 2 M GuanidiniumHCl 70%

thermofisher.com/PAGEsampleprep 40% Sucrose 70%

Sample preparation and Electrophoresis chamber Electrophoresis


For ordering information refer to page 85. Precast protein gels Protein standards Protein gel stains
electrophoresis buffers systems and power supplies run conditions
Prepare samples Choose the electrophoresis
28 Select precast gel and select buffers Select the standard chamber system and power supply Run the gel Stain the gel Post stain Protein gel electrophoresis technical handbook 29

Select buffers Recommended SDS-PAGE buffers and reagents


Sample buffer Other Running buffer

Buffers and reagents Gel type


optimized for use
with the gel
compatible
sample buffers
optimized for use
with the gel
Bolt Bis-Tris Bolt Sample Reducing Pierce LDS Bolt MES SDS MES vs. MOPS Running Buffer:

Protein samples prepared for If suitable, negatively charged, Plus Gel Agent (10X)
Bolt LDS Sample Buffer
Sample Buffer (4X)
for storage at RT
Running Buffer (20X)
Bolt MOPS SDS Running
Use MES SDS running buffers
to resolve small molecular
(4X) (nonreducing) Buffer (20X) weight proteins.
PAGE analysis are denatured low molecular weight dye is also Bolt Antioxidant Pierce Lane Use MOPS running buffers to resolve
Marker Non- mid-size proteins.
by heating in the presence of included in the sample buffer; NuPAGE NuPAGE Sample NuPAGE MES SDS

Reducing Sample
Bis-Tris Gel Reducing Agent (10X) Buffer (5X) Running Buffer (20X) MES has a lower pKa than MOPS,
NuPAGE Antioxidant NuPAGE MOPS SDS enabling gels with MES running buffer
a sample buffer with or without it will migrate at the buffer front, NuPAGE LDS Sample
storage at RT;
when you desire to Running Buffer (20X) to run faster than gels with MOPS SDS
Buffer (4X) (nonreducing) dilute your sample running buffer. The difference in ion
a reducing agent. The protein enabling one to monitor the less and require migration affects stacking and results in
a difference in protein separation range
transferable marker

sample is mixed with the sample progress of electrophoresis. dye to nitrocellulose


membranes
between these buffers.

buffer and heated for 210 The most common tracking dye NuPAGE
Tris-Acetate Gel
Novex Tris-Glycine SDS
Sample Buffer (2X)
Pierce Lane
NuPAGE Tris-Acetate SDS
Running Buffer (20X)
Reducing agent:
When preparing samples for
Marker Reducing
minutes, then cooled to room for sample loading buffers is NuPAGE Sample
Reducing Agent (10X)
Sample Buffer
(5X)when you
Novex Tris-Glycine Native
Running Buffer (10X)
reducing gel electrophoresis, any of the
following reducing agents may
Novex Tris-Glycine Native be used:
temperature before it is applied bromophenol blue. Sample Buffer (2X)
desire to dilute your
sample less and
B olt Sample Reducing Agent
require transferable N
 uPAGE Sample Reducing Agent
to the sample well on the gel. Novex Tris-Glycine Novex Tris-Glycine SDS
marker dye to
Novex Tris-Glycine SDS

We offer premixed, reliable Gel Sample Buffer (2X)


NuPAGE Sample nitrocellulose
Running Buffer (10X)
Novex Tris-Glycine Native
 ithiothreitol (DTT), 50 mM final
D

Loading buffers also contain Reducing Agent membranes Running Buffer (10X)
concentration

SDS-PAGE buffers and reagents Novex Tris-Glycine Native Pierce Tris-Glycine SDS -mercaptoethanol (-ME), 2.5%

glycerol so that they are heavier Sample Buffer (2X) Buffer (10X) final concentration
including sample buffers, BupH Tris-Glycine-SDS
Buffer Packs
tris(2-carboxyethyl)phosphine (TCEP),
than water and sink neatly to the 50 mM final concentration
running buffers, reducing agents, Novex Tricine Gel Novex Tricine SDS Sample Novex Tricine SDS Running
Add the reducing agent to the sample
bottom of the buffer-submerged Buffer (2X) Buffer (10X)
up to an hour before loading the gel.
and antioxidants. Avoid storing reduced samples for long

well when added to a gel. NativePAGE Gel NativePAGE Sample Buffer NativePAGE Running
periods, even if they are frozen.
Reoxidation of samples can occur
(4X) Buffer (20X)
during storage and produce
NativePAGE 5% G-250 NativePAGE Cathode
inconsistent results.
Sample Additive Buffer Additive (20X)

Novex IEF Gel Novex IEF Sample Buffer, Novex IEF Anode Buffer
pH 310 (2X) (50X)
Novex IEF Sample Buffer, Novex IEF Cathode Buffer,
pH 37 (2X) pH 310 (10X)
Novex IEF Cathode Buffer,
pH 37 (10X)

Novex Zymogram Novex Tris-Glycine SDS Novex Tris-Glycine SDS


Gels* Sample Buffer (2X) Running Buffer (10X)

*Novex Zymogram Developing Buffer (10X) and Novex Zymogram Renaturing Buffer (10X) are available for
visualizing the Novex Zymogram gels.
Learn more at
thermofisher.com/electrophoresisbuffers

Sample preparation and Electrophoresis chamber Electrophoresis


For ordering information refer to page 85. Precast protein gels Protein standards Protein gel stains
electrophoresis buffers systems and power supplies run conditions
Prepare samples Choose the electrophoresis
30 Select precast gel and select buffers Select the standard chamber system and power supply Run the gel Stain the gel Post stain Protein gel electrophoresis technical handbook 31

Buffer recipes
NuPAGE buffer recipes Tris-glycine buffer recipes
Buffer Storage Component Concentration (1X) Buffer Storage Component Concentration (1X)
NuPAGE LDS Sample Buffer +425C Tris-Glycine SDS Sample Buffer +4C
Glycerol 0% Tris HCl* 63 mM
Tris base 141 mM Glycerol 10%
Tris HCl 106 mM SDS 2%
LDS 2% Bromophenol Blue 0.0025%
EDTA 0.51 mM Deionized water
SERVA Blue G-250 0.22 mM (pH 6.8)
Phenol Red 0.175 mM
Tris-Glycine Native Sample Buffer +4C
(pH 8.5)
Tris HCl* 100 mM
NuPAGE MOPS SDS Running Buffer* +425C Glycerol 10%
MOPS 50 mM Bromophenol Blue 0.0025%
Tris base 50 mM Deionized water
SDS 0.1% (pH 8.6)
EDTA 1 mM
Tris-Glycine SDS Running Buffer Room
(pH 7.7)
temperature Tris base 25 mM
NuPAGE MES SDS Running Buffer* +425C Glycine 192 mM
MES 50 mM SDS 0.1%
Tris base 50 mM Deionized water
SDS 0.1% (pH 8.3)
EDTA 1 mM
Tris-Glycine Native Running Buffer Room
(pH 7.3)
temperature Tris base 25 mM
NuPAGE Transfer Buffer +425C Glycine 192 mM
Bicine 25 mM Deionized water
Bis-Tris (free base) 25 mM (pH 8.3)
EDTA 1.0 mM
Tris-Glycine Transfer Buffer Room
Chlorobutanol 0.05 mM
temperature Tris base 12 mM
(pH 7.2)
Glycine 96 mM
NuPAGE Tris-Acetate SDS Running Buffer +425C Deionized water
Tris base 50 mM (pH 8.3)
Tricine 50 mM
* Tris HCl solutions are prepared from Tris base and pH adjusted with 6 N HCl.
SDS 0.1%
(pH 8.24)

* The pre-mixed buffers (Cat. Nos. NP0001 and NP0002) also contain trace amounts of the proprietary NuPAGE Antioxidant (Cat. No. NP0005) for
stability. Additional Antioxidant may be required with specific protocols.

Sample preparation and Electrophoresis chamber Electrophoresis


For ordering information refer to page 85. Precast protein gels Protein standards Protein gel stains
electrophoresis buffers systems and power supplies run conditions
Prepare samples Choose the electrophoresis
32 Select precast gel and select buffers Select the standard chamber system and power supply Run the gel Stain the gel Post stain Protein gel electrophoresis technical handbook 33

Buffer recipes
Tricine buffer recipes Isoelectric focusing buffer recipes
Buffer Storage Component Concentration (1X) Buffer Storage Component Concentration (1X)
Tricine SDS Sample Buffer +4C IEF Sample Buffer pH 3-7 +4C
Tris HCl* 450 mM Lysine (free base) 40 mM
Glycerol 12%
SDS 4% Glycerol 15%
Coomassie Blue G 0.0075% Deionized water
Phenol Red 0.0025%
IEF Sample Buffer pH 3-10 +4C
Deionized water
Arginine (free base) 20 mM
(pH 8.45)
Lysine (free base) 20 mM
Tricine SDS Running Buffer Room Glycerol 15%
temperature Tris base 100 mM Deionized water
Tricine 100 mM
IEF Cathode Buffer pH 3-7 +4C
SDS 0.1%
(upper buffer chamber) Lysine (free base) 40 mM
Deionized water
Deionized water
(pH 8.3)

* Tris HCl solutions are prepared from Tris base and pH adjusted with 6 N HCl.
IEF Cathode Buffer pH 3-10 +4C
(upper buffer chamber) Arginine (free base) 20 mM
Lysine (free base) 20 mM
Deionized water
(pH 10.1)

Zymogram buffer recipes IEF Anode Buffer (for both pH ranges) Room
(lower buffer chamber) temperature Phosphoric acid 85% 7 mM
Buffer Storage Component Concentration (1X) Deionized water
(pH 2.4)
Zymogram Room
Renaturing Buffer temperature Triton X-100 2.7% (w/v) in H2O Urea-Thiourea-CHAPS 20C
Deionized water (rehydration buffer for IPG strips) Deionized urea 7M
Deionized thiourea 2M
Zymogram Room CHAPS 24%
Developing Buffer temperature Tris HCI* 50 mM Ampholytes* 0.22.0%
NaCl 200 mM Bromophenol Blue 0.002%
CaCl22 H2O 5 mM
Brij 35 0.006% (w/v) Ultrapure water
Deionized water _
(pH 7.6) DTT 20 mM
* Tris HCl solutions are prepared from Tris base and pH adjusted with 6 N HCl. * For ZOOM Strip pH 9-12 use 1% ZOOM Focusing Buffer pH 7-12 instead of ampholytes.

Sample preparation and Electrophoresis chamber Electrophoresis


For ordering information refer to page 85. Precast protein gels Protein standards Protein gel stains
electrophoresis buffers systems and power supplies run conditions
Prepare samples Choose the electrophoresis
34 Select precast gel and select buffers Select the standard chamber system and power supply Run the gel Stain the gel Post stain Protein gel electrophoresis technical handbook 35

Select the standard


Protein ladders and standards Unstained protein ladders
Ready-to-use prestained and unstained protein
ladders with exceptional lot-to-lot consistency
Low range PageRuler Unstained Low Range Protein Ladder

To assess the relative molecular each marker protein migrates Broad range PageRuler Unstained Protein Ladder We offer a broad range of prestained and unstained protein
ladders supplied in a ready-to-use format to facilitate easy protein
High range NativeMark Unstained Protein Standard

weights (sizes) of proteins in a through the gel. After measuring Recommended for:
analysis during gel electrophoresis and western blotting (Table 3).
Precise determination of target protein molecular weight All of our protein ladders offer:
sample, a mixture containing the migration distance that an Performancesharp protein bands and consistent migration

several proteins of known unknown protein travels through Prestained protein ladders
patterns provide easy molecular weight determination

Convenienceprotein ladders are ready to load, with no


molecular mass are run alongside the same gel, its molecular weight Low range PageRuler Prestained Protein Ladder heating required

Reliabilityexceptional lot-to-lot consistency


the test sample lane(s). Often can be determined graphically from Broad range PageRuler Plus Prestained Protein Ladder
Spectra Multicolor Broad Range Protein Ladder and reproducibility

these protein mixtures are run the standard curve. High range HiMark Prestained Protein Standard
Spectra Multicolor High Range Protein Ladder
Recommended for: Learn more at
on the outer lanes of the gel, to
Several kinds of ready-to-use Approximate determination of molecular weight
Monitoring the progress of electrophoresis runs
thermofisher.com/proteinstandards
maximize the number of remaining Estimating the efficiency of protein transfer to the membrane during
protein molecular weight (MW) western blotting

gel wells for test samples, but can


markers are available that are
also be useful in the middle wells Other
labeled, prestained, or unstained Western MagicMark XP Western Protein Standard
of the gel when running a large
for different modes of detection Specialty PageRuler Prestained NIR Protein Ladder
BenchMark Fluorescent Protein Standard
gel with many wells. Such sets of BenchMark His-tagged Protein Standard
and downstream applications. We IEF Marker 3-10
known protein mixtures are called
offer ladders suitable for both SDS-
protein molecular weight markers
PAGE as well as native PAGE.
or protein ladders. It is important
to choose a protein ladder that
consists of proteins with molecular
weights that span the molecular
weight range of the protein(s) of
interest. A standard curve can be
constructed from the distances

Sample preparation and Electrophoresis chamber Electrophoresis


Precast protein gels Protein standards Protein gel stains
electrophoresis buffers systems and power supplies run conditions
Prepare samples Choose the electrophoresis
36 Select precast gel and select buffers Select the standard chamber system and power supply Run the gel Stain the gel Post stain Protein gel electrophoresis technical handbook 37

Table 3. Protein standard selection guide


No. of Protein MW Protein band Monitoring Coomassie dye, silver, Monitoring protein Chemiluminescent
Category Product Range bands Reference bands determination visualization electrophoresis run or fluorescent staining transfer band visualization
Unstained ladders and standards
Unstained standards PageRuler Unstained Low 3.4100 kDa 8 25 kDa Best NA NA Best NA Good
Range Protein Ladder

PageRuler Unstained Protein 10200 kDa 14 50 kDa Good NA NA Good NA Good


Ladder

NativeMark Unstained Protein 201,200 kDa 8 Best for native NA NA Best NA Good
Standard electrophoresis

Pretained protein ladders


Prestained protein PageRuler Prestained Protein 10180 kDa 10 Green 10 kDa; orange Good Good Good NA Good Good
standards Ladder 70 kDa

PageRuler Plus Prestained 10250 kDa 9 Green 10 kDa; orange Good Good Good NA Good NA
Protein Ladder 25 and 70 kDa

HiMark Prestained Protein 30460 kDa 9 Best for high MW Good Good NA Best for high MW proteins NA
Standard proteins

Spectra Multicolor Broad Range 10260 kDa 10 Green 10 and 50 kDa; Good Best Best NA Best NA
Protein Ladder orange 40, 70, and
260 kDa; pink 140 kDa
Spectra Multicolor High Range 40300 kDa 8 Green 50 kDa; orange Good Best Best NA Best NA
Protein Ladder 70 and 300 kDa

Other ladders and standards


IEF IEF Marker 3-10 pI 3.510.7 13 Best for pI estimation NA NA Good NA NA

Chemiluminescent MagicMark XP Western Protein 20220 kDa 9 Good NA NA Good NA Best


standard Standard

Near infrared (NIR) PageRuler Prestained NIR 11250 kDa 10 55 kDa Good NA NA NA NA NA
standard Protein Ladder

Fluorescent BenchMark Fluorescent Protein 11155 kDa 7 Good NA NA NA NA Good


standard Standard

His-tag standard BenchMark His-tagged Protein 10160 kDa 10 Best NA NA Good NA Good for detection with
Standard anti-His antibody

Learn more at
thermofisher.com/proteinstandards

Sample preparation and Electrophoresis chamber Electrophoresis


For ordering information refer to page 86. Precast protein gels Protein standards Protein gel stains
electrophoresis buffers systems and power supplies run conditions
Prepare samples Choose the electrophoresis
38 Select precast gel and select buffers Select the standard chamber system and power supply Run the gel Stain the gel Post stain Protein gel electrophoresis technical handbook 39

Unstained ladders and standards

PageRuler Unstained Low PageRuler Unstained


Range Protein Ladder Protein Ladder
Sharp bands and precise molecular Sharp bands and precise molecular
weight estimation for low molecular weight estimation for a wide
weight proteins PageRuler Unstained Low Range Protein Ladder range of proteins PageRuler Unstained Protein Ladder
NuPAGE 412% Bis-Tris Gel with MES SDS buffer NuPAGE 412% Bis-Tris Gel with MES SDS buffer

Thermo Scientific PageRuler Unstained Low Thermo Scientific PageRuler Unstained Protein
Range Protein Ladder is a mixture of eight proteins Ladder is a mixture of 14 recombinant, highly
and peptides for use as size standards that resolve purified, unstained proteins for use as size
into clearly identifiable sharp bands when analyzed Storage specifications standards in SDS-PAGE and western blotting. Each Storage specifications
by SDS-PAGE. The proteins (except for the 5 and Storage buffer: Tris-H3PO4, EDTA, SDS, DTT, sodium azide,
protein in the ladder contains an integral Strep-tag Storage buffer: Tris-H3PO4, EDTA, SDS, DTT, sodium azide,
3.4 kDa peptides) contain an integral Strep-tag II bromophenol blue, and glycerol II Sequence, which can be detected directly on bromophenol blue, and glycerol
Sequence and may be detected on western blots western blots using a Strep-Tactin Conjugate or an
Storage conditions: upon receipt store at 20C Storage conditions: upon receipt store at 20C
using Strep-Tactin Conjugates. antibody against the Strep-tag II Sequence.
Stability: 1 year from date of receipt Stability: 1 year from date of receipt
Comprehensiveeight proteins and peptides spanning Comprehensive14 highly purified proteins with excellent
3.4 to 100 kDa; the 25 kDa band is more intense than the accuracy spanning 10 to 200 kDa; the ladder contains one
Recommended products Recommended products
other bands for easy orientation 50 kDa reference band of higher intensity
The PageRuler Unstained Protein Ladder is recommended for Novex The PageRuler Unstained Protein Ladder is recommended for Novex
Versatilecompatible with western blots by staining with Tris-Glycine, Bis-Tris or Tris-Acetate gels. Versatilecompatible with Coomassie dye; compatible Tris-Glycine, Bis-Tris or Tris-Acetate gels.
Ponceau S dye or Coomassie dye; compatible with Thermo with Pierce Reversible Protein Stain Kit for Nitrocellulose
Scientific Pierce Reversible Protein Stain Kit for Nitrocellu- Membranes, silver staining, or western blotting
lose Membranes or other protein stains
Learn more at
thermofisher.com/unstainedstandards Learn more at
thermofisher.com/unstainedstandards

Sample preparation and Electrophoresis chamber Electrophoresis


For ordering information refer to page 86. Precast protein gels Protein standards Protein gel stains
electrophoresis buffers systems and power supplies run conditions
Prepare samples Choose the electrophoresis
40 Select precast gel and select buffers Select the standard chamber system and power supply Run the gel Stain the gel Post stain Protein gel electrophoresis technical handbook 41

Prestained ladders

NativeMark Unstained kDa kDa kDa kDa


1,2361,236
PageRuler Prestained
Protein Standard Protein Ladder
1,0481,048

1,2361,236
1,0481,048 720 720

480 480

Convenient molecular weight estimation 720 720


242 242
Outstanding clarity for easy molecular
for native electrophoresis 480 480
146 146 NativeMark Unstained weight determination of low molecular
242 242
66 66
Protein Standard
NativePAGE Bis-Tris gels
weight proteins PageRuler Prestained Protein Ladder
146 146
The Invitrogen NativeMark Unstained
NuPAGE 412% Bis-Tris Gel with MES SDS buffer
66 66
Protein Standard is designed for molecular Thermo Scientific PageRuler Prestained Protein
20 20
weight estimation of proteins using native gel 20 20 Ladder is a mixture of 10 blue-, orange-, and
electrophoresis. 312%
312% 416%
416% green-stained proteins for use as size standards
in SDS-PAGE and western blotting. The mobility Storage specifications
Comprehensivecontains a wide range of high molecular
Storage specifications of prestained proteins can vary in different SDS- Storage buffer: Tris-H3PO4, EDTA, SDS, DTT, sodium azide,
weight proteins, providing 8 protein bands in the range
Storage buffer: Bis/Tris-HCl (pH 7.0), NaCl, glycerol, and
PAGE buffer systems; however, they are suitable for bromophenol blue, and glycerol
of 201,200 kDa
Ponceau S approximate molecular weight determination when
Storage conditions: upon receipt store at 20C
Versatilecan be visualized using Coomassie, silver, or calibrated against unstained standards in the
fluorescent stains after electrophoresis, or with Ponceau S, Storage conditions: upon receipt store at 20C
same system. Stability: 1 year from date of receipt
Coomassie, or other membrane stains after western transfer Stability: 6 months
Comprehensivecontains 10 proteins with a range of 10
Recommended products
to 180 kDa; includes one 70 kDa reference protein colored
Recommended products The PageRuler Prestained Protein Ladder is recommended for use with
with an orange dye and one 10 kDa reference protein
The NativeMark Unstained Protein Standard is recommended for use Tris-glycine, Bis-Tris, and Tris-acetate gels.
colored with a green dye
with NativePAGE Bis-Tris gels, Novex Tris-Glycine gels, or NuPAGE
Tris-Acetate gels. Versatilecompatible with Coomassie dye staining and
western blotting
Learn more at
Learn more at thermofisher.com/prestainedstandards
thermofisher.com/unstainedstandards

Sample preparation and Electrophoresis chamber Electrophoresis


For ordering information refer to page 86. Precast protein gels Protein standards Protein gel stains
electrophoresis buffers systems and power supplies run conditions
Prepare samples Choose the electrophoresis
42 Select precast gel and select buffers Select the standard chamber system and power supply Run the gel Stain the gel Post stain Protein gel electrophoresis technical handbook 43

PageRuler Plus Prestained HiMark Prestained kDa kDa


460
kDa kDa

Protein Ladder Protein Standard


198 220
268 260
98 238 160
120
100 110
62 80 80
171

Outstanding clarity for easy molecular Superb analysis of high molecular


60
49 60 50
117 40

weight determination of a broad weight proteins


50
38
30
40
PageRuler Plus Prestained Protein Ladder 28

range of proteins NuPAGE 412% Bis-Tris Gel with MES SDS buffer 17
71
15
20

14 HiMark Prestained
30
Protein Standard
The Invitrogen HiMark Prestained Protein
55
NuPAGE 38%10Tris-acetate SDS buffer
Thermo Scientific PageRuler Plus Prestained Standard is designed for analysis of high molecular6 41
20
3.5

Protein Ladder is a mixture of 9 blue-, orange-, and weight proteins on NuPAGE Tris-acetate gels. 3
31

Standard SeeBlue HiMark MagicMark XP Novex Sharp


green-stained proteins for use as size standards in Plus2 Pre-stained Pre-stained
Comprehensivecontains a wide range of high molecular
SDS-PAGE and western blotting. The mobility of Cat. No. LC5925 LC5699 LC5602 LC5800
weight proteins, providing 9 protein bands in the range of
prestained proteins can vary in different SDS-PAGE Storage specifications
NuPAGE
Storage
Tris-Acetatespecifications
NuPAGE 38% NuPAGE Bis-Tris Gel, NuPAGE

412% Bis-Tris Gel blotted to 412% Bis-Tris


30460 kDa Gel w/ MES w/ Tris-acetate nitrocellulose, Gel w/ MES
buffer systems; however, they are suitable Storage buffer: Tris-H3PO4, EDTA, SDS, DTT, sodium azide,
SDS buffer SDS buffer
Storage buffer:
detected w/ SDS buffer
Tris-HCl, formamide, SDS, and phenol red
WesternBreeze

Chemiluminescent Kit
for approximate molecular weight determination bromophenol blue, and glycerol
Versatileeasy visualization of band migration during
when calibrated against unstained standards in electrophoresis and rapid evaluation of western Storage conditions: upon receipt store at 20C
Storage conditions: upon receipt store at 20C transfer efficiency
the same system. Stability: 6 months from date of receipt
Stability: 1 year from date of receipt
Comprehensive9 proteins with a broad range of 10 to
Recommended products
250 kDa; includes 70 kDa and 25 kDa reference proteins
Recommended products The HiMark Prestained Protein Standard is recommended for use with
that are colored with an orange dye and one 10 kDa
The PageRuler Plus Prestained Protein Ladder is recommended for Tris- NuPAGE Tris-Acetate gels under denaturing conditions. This standard
reference protein that is colored with a green dye
glycine, Bis-Tris, and Tris-acetate gels. can also be used with NuPAGE 412% Bis-Tris gels with Invitrogen
Versatilecompatible with Coomassie dye staining and NuPAGE MOPS SDS Running Buffer and Novex 4% Tris-Glycine gels.
However, to obtain the best results with high molecular weight proteins,
western blotting
always use NuPAGE Tris-Acetate gels.
Learn more at
The HiMark Prestained Protein Standard is also available as part of the
thermofisher.com/prestainedstandards
following kits that include gels, running and sample buffers, and stains or
blotting materials:
Invitrogen NuPAGE Large Protein Staining Kit
Invitrogen NuPAGE Large Protein Sensitive Staining Kit
Invitrogen NuPAGE Large Protein Blotting Kit

Learn more at
thermofisher.com/prestainedstandards

Sample preparation and Electrophoresis chamber Electrophoresis


For ordering information refer to page 86. Precast protein gels Protein standards Protein gel stains
electrophoresis buffers systems and power supplies run conditions
Prepare samples Choose the electrophoresis
44 Select precast gel and select buffers Select the standard chamber system and power supply Run the gel Stain the gel Post stain Protein gel electrophoresis technical handbook 45

Spectra Multicolor Broad Spectra Multicolor High


Range Protein Ladder Range Protein Ladder
Superior visualization and analysis of a Superior and convenient visualization of
broad range of proteins Spectra Multicolor Broad Range Protein Ladder high molecular weight proteins
NuPAGE 412% Bis-Tris Gel with MES SDS buffer
Spectra Multicolor High Range Protein Ladder
NuPAGE 412% Bis-Tris Gel with MES SDS buffer
Thermo Scientific Spectra Multicolor Broad

Thermo Scientific Spectra Multicolor High Range

Range Protein Ladder is a 4-color protein standard Protein Ladder is a mixture of 8 blue-, green-,
containing 10 prestained proteins for use in gel and orange-stained proteins for use as size
electrophoresis and western blotting. This standard standards for high molecular weight proteins in gel
is designed for monitoring the progress of gels electrophoresis and western blotting. This marker Storage specifications
during SDS-PAGE and for assessing western blot Storage specifications is designed for monitoring the progress of gels Storage buffer: Tris-H3PO4, EDTA, SDS, DTT, sodium azide,
transfer efficiency. Four different chromophores Storage buffer: Tris-H3PO4, EDTA, SDS, DTT, sodium azide,
during SDS-PAGE, assessing western blot transfer bromophenol blue, and glycerol
(blue, orange, green, and pink) are bound to the bromophenol blue, and glycerol efficiency, and estimating the approximate size of
Storage conditions: upon receipt store at 20C
different component proteins, producing a brightly proteins after gel staining or western blotting.
Storage conditions: upon receipt store at 20C
colored ladder with an easy-to-remember pattern. Stability: 1 year from date of receipt
Comprehensive8 proteins of similar intensity spanning
Stability: 1 year from date of receipt
Comprehensive10 proteins with similar intensity span- a range of 40 to 300 kDa; 3 different chromophores (blue,
Recommended products
ning a broad range of 10 to 260 kDa orange, and green) are bound to the different component
Recommended products The Spectra Multicolor High Range Protein Ladder is recommended for
proteins, producing a brightly colored ladder with an
Versatilecompatible with Coomassie dye staining and The Spectra Multicolor Broad Range Protein Ladder is Tris-glycine, Bis-Tris, and Tris-acetate gels.
easy-to-remember pattern
western blotting recommended for Tris-glycine, Bis-Tris, and
Tris-acetate gels. Versatilecompatible with Coomassie dye staining and
western blotting

Learn more at Learn more at


thermofisher.com/prestainedstandards thermofisher.com/prestainedstandards

Sample preparation and Electrophoresis chamber Electrophoresis


For ordering information refer to page 86. Precast protein gels Protein standards Protein gel stains
electrophoresis buffers systems and power supplies run conditions
Prepare samples Choose the electrophoresis
46 Select precast gel and select buffers Select the standard chamber system and power supply Run the gel Stain the gel Post stain Protein gel electrophoresis technical handbook 47

Other ladders and standards

MagicMark XP Western kDa kDa


460
kDa kDa
PageRuler Prestained NIR
Protein Standard Protein Ladder
198 220
268 260
98 238 160
120
100 110
62 80 80
171
60

Accurate molecular weight estimation 49


117
60

50
50
40
Sharp prestained standard for near-IR
directly on western blots fluorescent visualization and protein sizing
38
30
40
28
MagicMark
20
XP Western Protein Standard
71
17 NuPAGE Bis-Tris gel, blotted to nitrocellulose, PageRuler Prestained NIR Protein Ladder
30 15
14
55 and detected with Invitrogen WesternBreeze NuPAGE 412% Bis-Tris Gel with MES
The MagicMark XP Western Protein Standard 10
Chemiluminescent Kit Thermo Scientific PageRuler Prestained NIR SDS buffer
6 20
is specifically designed for easy and convenient 41 3.5
Protein Ladder is a mixture of 10 proteins that
3
protein molecular weight estimation directly on 31 are stained blue and labeled with a fluorophore Visual Infrared
Standard SeeBlue HiMark
MagicMark XP Novex Sharp

western blots. Each recombinant protein Plus2 in the Pre-stained Pre-stained for near-infrared (NIR) fluorescent visualization detection detection

standard contains an IgG binding site, which


Cat. No. LC5925 LC5699 LC5602
Storage specifications LC5800
and protein sizing following electrophoresis. The
NuPAGE NuPAGE 38%

NuPAGE Bis-Tris Gel,

NuPAGE

binds the primary or secondary antibody 412%used


Bis-Tris
Gel w/ MES
Tris-Acetate Gel
w/ Tris-acetate 
S
blotted to
torage
nitrocellulose,
412% Bis-Tris
buffer: Tris-HCl
Gel w/ MES (pH 6.8), DTT, glycerol, SDS, and
molecular weight markers in this ladder resolve into Storage specifications
for detection of the target protein, allowing direct SDS buffer Chemiluminescent
SDS buffer detected w/

bromophenol
WesternBreeze
Kit

SDS buffer

blue sharp bands when analyzed by SDS-PAGE. The Storage buffer: Tris-H3PO4, EDTA, SDS, DTT, sodium azide,
visualization of the standard on the western blot. 55 kDa band is of greater intensity and serves as a bromophenol blue, and glycerol
Storage conditions: upon receipt store at 20C
reference band.
Comprehensiveconsists of 9 recombinant proteins from Storage conditions: upon receipt store at 20C
Stability: 4 months from date of receipt
20 to 220 kDa Comprehensive10 protein bands spanning 11 to
Stability: 1 year from date of receipt
250 kDa
Versatilecompatible with chemiluminescent, Recommended products
chromogenic, and fluorescent detection The MagicMark XP Western Protein Standard is compatible Versatilevisualize using instruments equipped for Recommended products
with a broad range of gelsNuPAGE Bis-Tris gels, detection of near-infrared fluorescence such as certain The PageRuler Prestained NIR Protein Ladder is recommended for visual
Novex Tris-Glycine gels, Novex Tricine gels, NuPAGE Typhoon Imagers and the LI-COR Odyssey Infrared detection, infrared imaging detection, and western blotting.
Tris-Acetate gels, and Bolt Bis-Tris Plus gels. Imaging System; bands are directly visible because the
proteins are prestained blue

Learn more at Learn more at


thermofisher.com/westernblotstandard thermofisher.com/specialtystandards

Sample preparation and Electrophoresis chamber Electrophoresis


For ordering information refer to page 86. Precast protein gels Protein standards Protein gel stains
electrophoresis buffers systems and power supplies run conditions
Prepare samples Choose the electrophoresis
48 Select precast gel and select buffers Select the standard chamber system and power supply Run the gel Stain the gel Post stain Protein gel electrophoresis technical handbook 49

BenchMark Fluorescent BenchMark His-tagged


Protein Standard Protein Standard
Efficient estimation of molecular weight by Convenient detection and protein sizing
fluorescent detection BenchMark Fluorescent Protein Standard
of His-tagged proteins
NuPAGE 412% Bis-Tris Gel with MES SDS buffer BenchMark His-tagged Protein Standard
NuPAGE 412% Bis-Tris Gel with MES
The Invitrogen BenchMark Fluorescent Protein

The Invitrogen BenchMark His-tagged Protein

SDS buffer
Standard consists of Alexa Fluor 488 dye Standard can be used as a positive control and for
conjugated proteins for molecular weight estimation molecular weight sizing in His-tagged fusion protein SimplyBlue InVision
stain stain
of fluorescently labeled proteins. detection. Each protein in the standard has a 6xHis
Storage specifications tag. Storage specifications
Comprehensiveconsists of 7 distinct protein bands in the
Storage buffer: Tris-HCl, SDS, glycerol, and Coomassie Storage buffer: Tris-HCl, SDS, glycerol, DTT, and Coomassie
range of ~11155 kDa Comprehensive10 sharp and clear bands from 10 to 160
Blue G-250 Blue G-250
kDa for molecular weight estimation of His-tagged proteins
Versatilevisualize on a UV transilluminator or laser-based
Storage conditions: upon receipt store at 20C Storage conditions: upon receipt store at 20C
scanning instrument after SDS-PAGE Versatilecan be visualized with Invitrogen InVision His-
Stability: 6 months from date of receipt Tag In-Gel Stain or Coomassie R-250 stain on SDS-PAGE Stability: 6 months from date of receipt
gels, or with Anti-His (C-term) Antibody using chromogenic
Recommended products or chemiluminescent detection systems Recommended products
The BenchMark Fluorescent Protein Standard is recommended for use The BenchMark His-tagged Protein Standard is recommended for use with
with NuPAGE gels or Novex Tris-Glycine gels. Learn more at NuPAGE gels and Novex Tris-Glycine gels.
thermofisher.com/specialtystandards

Learn more at
thermofisher.com/specialtystandards IEF Marker 3-10
Accurate determination of protein
isoelectric points

The IEF Marker 3-10 is a ready-to-use protein IEF Marker 3-10


Novex pH 310 Gel
standard developed for IEF applications. This
marker can be used for monitoring of protein
separation on IEF gels and pI determination of
unknown protein samples.
Storage specifications
Comprehensive13 purified isoforms from pI 3.510.7;
no additional high range or low range markers are required Storage buffer: 10% glycerol containing bromophenol
blue (0.01%) and methyl red (0.01%)
Versatilecan be used for both native and
denaturing conditions Storage conditions: upon receipt store at 20C

Stability: 1 year from date of receipt

Learn more at
thermofisher.com/iefstandards Recommended products
The IEF Marker 3-10 is applicable to all IEF gels (vertical or horizontal).

Sample preparation and Electrophoresis chamber Electrophoresis


For ordering information refer to page 86. Precast protein gels Protein standards Protein gel stains
electrophoresis buffers systems and power supplies run conditions
Prepare samples Choose the electrophoresis
50 Select precast gel and select buffers Select the standard chamber system and power supply Run the gel Stain the gel Post stain Protein gel electrophoresis technical handbook 51

Electrophoresis chambers
and power supplies
Electrophoresis run considerations:

In electrical terms, the process of


electrophoresis is closely
associated with the following
equations derived from Ohms Law:
Voltage = Current Resistance (V = IR)
Which electrophoresis chamber system is right for you?
Wattage = Current Voltage (W = IV)
Mini Gel Tank XCell SureLock Mini-Cell XCell4 SureLock Midi-Cell

Resistance Current
The electrical resistance of the assembled electrophoresis cell For a given gel/buffer system, at a given temperature, current
is dependent on buffer conductivity, gel thickness, temperature, varies in proportion to the field strength (voltage) and cross-
and the number of gels being run. Although the resistance is sectional area (thickness and number of gels). When using a
determined by the gel system, the resistance varies over the constant current setting, migration starts slow, and accelerates
course of the run. over time, thus favoring stacking in discontinuous gels.

In discontinuous buffer systems (and to a lesser extent in When running under constant current, set a voltage limit on the
continuous buffer systems) resistance increases over the course power supply at, or slightly above the maximum expected voltage
of electrophoresis. This occurs in the Tris-glycine buffer system to avoid unsafe conditions. At constant current voltage increases
as highly conductive chloride ions in the gel are replaced by less as resistance increases. If a local fault condition occurs (e.g., a Gel capacity Up to 2 minigels Up to 2 minigels (8 x 8 cm) Up to 4 midigels (8 x 13 cm)
conductive glycine ions from the running buffer. bad connection), high local resistance may cause the voltage to
Cell dimensions 32 x 11.5 x 16 cm 14 x 13 x 16 cm 21 x 19 x 16 cm
reach the maximum for the power supply, leading to overheating (L x W x H) (height with lid on) (height with lid on) (height with lid on)
Resistance decreases as the temperature increases.
and damage of the electrophoresis cell.
Advantages The Mini Gel Tank is versatile and XCell II Blot Module is available for Advanced apparatus for easier,
compatible with NuPAGE, Bolt, or semi-wet protein transfers more reliable electrophoresis with
Voltage Power Novex minigels. The unique tank Instrument incorporates a gel midigels
design enables convenient side- tension wedge in place of the rear
The velocity of an ion in an electric field varies in proportion to the by-side gel loading and enhanced wedge used on earlier models
Wattage measures the rate of energy conversion, which is
field strength (volts per unit distance). The higher the voltage, the viewing during use.
manifested as heat generated by the system. Using constant Mini Blot Module is available for wet
faster an ion moves. For most applications, we recommend a
power ensures that the total amount of heat generated by the protein transfers.
constant voltage setting.
system remains constant throughout the run, but results in variable
A constant voltage setting allows the current and power to mobility since voltage increases and current decreases over the
decrease over the course of electrophoresis, providing a safety course of the run. Constant power is typically used when using Learn more at
margin in case of a break in the system. IEF strips. When using constant power, set the voltage limit slightly thermofisher.com/electrophoresischambers
above the maximum expected for the run. High local resistance
The constant voltage setting does not need adjustment to can cause a large amount of heat to be generated over a small
account for differences in number or thickness of gels being distance, damaging the electrophoresis cell and gels.
electrophoresed.

Sample preparation and Electrophoresis chamber Electrophoresis


For ordering information refer to page 87. Precast protein gels Protein standards Protein gel stains
electrophoresis buffers systems and power supplies run conditions
Prepare samples Choose the electrophoresis
52 Select precast gel and select buffers Select the standard chamber system and power supply Run the gel Stain the gel Post stain Protein gel electrophoresis technical handbook 53

Mini Gel Tank


One tank, 181 gels
The Mini Gel Tank is designed for more intuitive use
and greater convenience compared to traditional
electrophoresis tanks (Figure 24). The unique,
side-by-side tank design allows you to perform
1. Snap the electrophoresis tank into the base, and 2. Remove the comb, and peel away the tape at the 3. Place the cassette in the chamber with the wells
electrophoresis of 1 or 2 minigels. place the cassette clamp(s) into the chamber(s) bottom of the gel cassette. facing towards you.
with the anode connector(s) (+) aligned to the center. Rinse the wells 3 times with 1X buffer. Hold the cassette in a raised position and close
Fill the chamber(s) with 1X buffer to the level of the clamp by moving the cam handle forward.
The Mini Gel Tank offers: the cathode.

Versatilitycompatible with all of our minigels, including


NuPAGE, Novex, Bolt, and specialty gels

Easy sample loadingforward-facing well configuration

Simultaneous visualization of both gelsstreamlined, Specifications


side-by-side tank configuration Gel capacity: up to 2 minigels
Simple monitoring of gelswhite tank stand provides Cell size (L x W x H): 32 x 11.5 x 16 cm (height with lid on)
easy visualization of prestained markers
Buffer requirement: 400 mL for each minigel chamber
4. Make sure the wells are completely filled with 5. Hold the cassette and release the cassette clamp. 6. Make sure the power supply is off.
Less running buffer requiredgel chambers are 1X buffer.
Material: polycarbonate Gently lower the casette so that it rests on the If only running one gel, remove the cassette
separated, so you only need to load sufficient buffer for Load your samples and markers. bottom of the chamber, and close the cassette clamp. clamp from unused chamber.
each gel to the specified fill line Chemical resistance: not compatible with acetone, chlorinated Add 1X buffer to the level of the fill line. Place the lid on the tank and plug the
hydrocarbons, or aromatic hydrocarbons electrode cords into the power supply.
Turn the power supply on to begin electrophoresis.
Figure 24. How to use the Mini Gel Tank.

Figure 25. Electrophoresis of Bolt gel using the Mini Gel Tank. Protein
standards and samples were loaded at 10 L sample volumes in an
Invitrogen Bolt 412% Bis-Tris Plus Gel. Electrophoresis was performed
using the Mini Gel Tank at 200 V (constant). Sharp, straight bands with
consistent migration patterns were observed after staining with SimplyBlue
SafeStain. Images were acquired using a flatbed scanner. Lane 1: SeeBlue
Plus2 Prestained Standard; Lane 2: 10 g E. coli lysate; Lane 3: Mark12
Unstained Standard (blend of 12 purified proteins); Lane 4: 40 g HeLa cell
lysate; Lane 5: 20 g HeLa cell lysate; Lane 6: 5 g BSA; Lane 7: 40 g
Jurkat cell lysate; Lane 8: 5 g GST fusion protein; Lane 9: Novex Sharp
Watch our Mini Gel Tank video. thermofisher.com/minigeltank Unstained Protein Standard; Lane 10: 5 g -galactosidase.

Recommended products
The Mini Blot Module is a wet transfer device that conveniently fits into
the chambers of the Mini Gel Tank to easily transfer proteins from minigels
to nitrocellulose or PVDF membranes.

Learn more at thermofisher.com/minigeltank

Sample preparation and Electrophoresis chamber Electrophoresis


For ordering information refer to page 87. Precast protein gels Protein standards Protein gel stains
electrophoresis buffers systems and power supplies run conditions
Prepare samples Choose the electrophoresis
54 Select precast gel and select buffers Select the standard chamber system and power supply Run the gel Stain the gel Post stain Protein gel electrophoresis technical handbook 55

XCell SureLock Mini-Cell Figure 26. How to use the


XCell SureLock Mini-Cell.

Simultaneous electrophoresis of up
to 2 minigels

The unique design of the Invitrogen XCell


SureLock Mini-Cell allows you to run minigels 1.Drop buffer core into the lower 2.Lock the gel tension wedge in 3.Place the cell lid on the unit
quickly and easily without any clamps or grease buffer chamber of the XCell place, load samples, and fill the and youre ready to run.
SureLock Mini-Cell. Insert one buffer chambers with the
(Figure 26). The tight seal provided by the gel minigel in front of the buffer core and appropriate running buffers.
tension wedge results in leak-free, consistent a second minigel or the buffer dam
behind the buffer core.
performance. The XCell SureLock Mini-Cell is
compatible with NuPAGE, Novex, and specialty
gels (Figure 27).
Figure 27. Electrophoresis of NuPAGE Bis-Tris gels with
the XCell SureLock Mini-Cell. Lane 1: SeeBlue Plus2
Key features of the XCell SureLock Mini-Cell: Specifications Prestained Standard; Lane 2: 10 g E. coli lysate; Lane 3:
Mark12 Unstained Standard (blend of 12 purified proteins);
User-friendly designuses single gel tension wedge with Gel capacity: up to 2 minigels Lane 4: 40 g HeLa cell lysate; Lane 5: 20 g HeLa cell lysate;
no clamps or grease Lane 6: not used; Lane 7: 40 g Jurkat cell lysate; Lane 8:
Cell size (L x W x H): 14 x 13 x 16 cm (height with lid on) 5 g of a GST fusion protein; Lane 9: Invitrogen Novex Sharp
Flexibilityperform electrophoresis of 2 minigels Protein Standard; and Lane 10: 5 g -galactosidase. Gel
Buffer chamber requirement (Novex minigels): electrophoresis was performed at 200 V (constant) and gels
simultaneously
were stained using SimplyBlue SafeStain. Images were acquired
Upper buffer chamber: 200 mL
Unique, heat dissipating designno need for a using a flatbed scanner.
Lower buffer chamber: 600 mL
cooling device NuPAGE Bis-Tris gel in
Chemical resistance: The XCell SureLock Mini-Cell is XCell SureLock Mini-Cell
Built-in safety featuresretractable plugs, recessed jacks,
impervious to most alcohols but not compatible with acetone,
and a specially designed lid enhances user safety
chlorinated hydrocarbons (e.g., chloroform), or aromatic
hydrocarbons (e.g., toluene, benzene)
Recommended products
The XCell SureLock Mini-Cell can be easily adapted for transfer of proteins
from minigels to membranes by simply inserting the XCell II Blot Module
in the lower buffer chamber.
Learn more at thermofisher.com/surelockmini

Sample preparation and Electrophoresis chamber Electrophoresis


For ordering information refer to page 87. Precast protein gels Protein standards Protein gel stains
electrophoresis buffers systems and power supplies run conditions
Prepare samples Choose the electrophoresis
56 Select precast gel and select buffers Select the standard chamber system and power supply Run the gel Stain the gel Post stain Protein gel electrophoresis technical handbook 57

XCell4 SureLock Midi-Cell A. 1. Insert the XCell4 SureLock Assembly in its unlocked
position into the center of the Midi-Cell base. The
XCell4 SureLock Assembly slides down over the
4. The upper buffer chamber (cathode) is the void
formed between a gel and the buffer core at the
center of each core.
protrusion in the Midi-Cell base.

Simultaneous electrophoresis of up to
4 midigels 2. Place one cassette on each side of the buffer core
for each of the two cores. For each cassette, the
5. Lock the XCell4 SureLock Assembly by moving the
tension lever to the locked position (indicated on the
shorter well side of the cassette must face out XCell4 SureLock Assembly). This will squeeze the
towards the lower buffer chamber. gels and buffer cores together, creating leak-free
seals.
The Invitrogen XCell4 SureLock Midi-Cell
allows simultaneous vertical electrophoresis of
14 midigels without leaking, enabling consistent B. 3. While holding the assembly together with your 6. Proceed to loading samples and buffers.
hands (A), insert the buffer cores with the gel
performance. The system is designed to dissipate cassettes into the lower buffer chamber such that
the negative electrode fits into the opening in the
heat effectively and evenly, and enable high- gold plate on the lower buffer chamber (B). Always
hold the cassette assembly by its edges as shown
resolution results when using Novex midigels in the figure.
(Figure 29). Note: If you are having difficulty inserting the
Specifications assembly into the lower buffer chamber, make sure
the cathode (black polarity indicator) of the buffer
Key features of the XCell4 SureLock Midi-Cell: Gel capacity: up to 4 midigels (8 x 13 cm) core is aligned with the cathode (black polarity
indicator) of the lower buffer chamber.
User-friendly designleak-free electrophoresis without Cell size (L x W x H): 21 x 19 x 16 cm (height with lid on)
clamps or grease Figure 28. How to use the XCell4 SureLock Midi-Cell with 4 gels.
Buffer chamber requirement:
Flexibilityperform electrophoresis of 14 midigels Upper buffer chamber: 175 mL x 4
Unique, heat dissipating designno need for a Lower buffer chamber: 540700 mL
cooling device Chemical resistance: not compatible with acetone, chlorinated 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
Figure 29. Quality of a precast NuPAGE Novex 412% Bis-Tris Midi
hydrocarbons, or aromatic hydrocarbons Gel with a variety of protein standards, lysates and purified proteins.
Built-in safety featuresspecially designed lid enhances
Electrophoresis was performed using MES running buffer and an XCell4
user safety SureLock Midi Cell at 200 V (constant). Following electrophoresis, the
gel was stained using SimplyBlue SafeStain, destained using water, and
imaged using a flatbed scanner. Sharp, straight bands were observed.
Lanes 1, 10, 11, and 20 were each loaded with 5 L of Mark12 Unstained
Standard (blend of 12 purified proteins). Lanes 2, 9, 12, and 19 were each
loaded with 10 g of E. coli lysate. Lanes 3 and 18 were each loaded
with 6 g of human IgG. Lanes 4 and 17 were each loaded with 6 g of
human IgM. Lanes 5 and 16 were each loaded with 5 L of SeeBlue Plus2
Prestained Protein Standard. Lanes 6 and 15 were each loaded with 5 L
of BenchMark Protein Ladder. Lanes 7 and 14 were each loaded with
15 L of MagicMark XP Western Protein Standard. Lanes 8 and 13 were
each loaded with 5 L of HiMark Unstained Protein Standard.

Learn more at thermofisher.com/surelockmidi

Sample preparation and Electrophoresis chamber Electrophoresis


For ordering information refer to page 87. Precast protein gels Protein standards Protein gel stains
electrophoresis buffers systems and power supplies run conditions
Prepare samples Choose the electrophoresis Prepare samples Choose the electrophoresis
58 Select precast gel and select buffers Select the standard chamber system and power supply Run the gel Stain the gel Post stain Select precast gel and select buffers Select the standard chamber system and power supply Run the gel Stain the gel Post stain 59

Run the gel


PowerEase 90W Table 4. Gel running conditions in electrophoresis chamber systems.

Running conditions in XCell Surelock Mini-Cell Running conditions in Mini Gel Tank
Power Supply Starting
current
End
current
Approximate
run time
Starting
current
Approximate
End current run time
Voltage (V) (mA)* (mA)* (minutes) Voltage (V) (mA)* (mA)* (minutes)
Simple, affordable power supply Bolt 412% (MES) NA NA NA NA 200 160 70 20
specifically for minigel electrophoresis Bolt 412% (MOPS) NA NA NA NA 200 160 50 35
NuPAGE 412% Bis-Tris 200 100 to 125 60 to 80 35 200 160 90 30
(MES)

The Invitrogen PowerEase 90W Power Supply NuPAGE 412% Bis-Tris 200 100 to 125 60 to 80 50 200 140 50 42
(MOPS)
is designed specifically for minigel electrophoresis.
Novex 420% Tris-Glycine 125 30 to 40 8 to 12 90 125 40 10 100
The straightforward, intuitive interface makes the (denatured)
powering of gel runs a simple and easy process. Novex 420% Tris-Glycine 125 6 to 12 3 to 6 1 to 12 125 30 10 90
In addition, the PowerEase 90W Power Supply (native) hours
features: NuPAGE 38% Tris-Acetate 150 40 to 55 25 to 40 60 150 60 20 50
(denatured)
Constant voltage or current settings NuPAGE 38% Tris-Acetate 150 18 7 2 to 3 hours 150 40 10 100
(native)
Built-in timer for walk-away gel electrophoresis Novex 1020% Tricine 125 80 40 90 125 110 40 65

Output jacks that are compatible with most NativePAGE 312% 150 12 to 16 2 to 4 90 to 115 150 10 <10 80

electrophoresis devices pH 3-10 IEF 100 7 NA 60 100 8 NA 60


200 NA NA 60 200 NA NA 60
500 NA 5 30 500 NA 5 30
10% Zymogram (Gelatin) 125 30 to 40 8 to 12 90 125 40 10 90
PowerEase 300W * Per gel.
Note: Run times may vary depending on the power supply and gel percentage.

Power Supply Learn more at thermofisher.com/powerease

Programmable power supply designed


for high-throughput gel electrophoresis

The Invitrogen PowerEase 300W Power Supply


is a fully programmable power supply designed
for high-throughput gel electrophoresis. The
straightforward, intuitive interface makes the
powering of gel runs a simple and easy process.
In addition, the PowerEase 300W Power Supply
features:

Constant voltage, current, or power settings

Built-in timer for walk-away gel electrophoresis

Up to 10 custom programs with 10 steps each

Four sets of output jacks that are compatible


with most electrophoresis devices

Sample preparation and Electrophoresis chamber Electrophoresis


For ordering information refer to page 87. Precast protein gels Protein standards Protein gel stains
electrophoresis buffers systems and power supplies run conditions
Prepare samples Choose the electrophoresis
60 Select precast gel and select buffers Select the standard chamber system and power supply Run the gel Stain the gel Post stain Protein gel electrophoresis technical handbook 61

Troubleshooting tips
XCell SureLock Mini-Cell troubleshooting Electrophoresis troubleshooting
Observation Cause Solution Problem Possible cause Suggested solution
Run taking longer Buffers are too dilute Check if buffer was diluted properly. Check buffer recipe; Run taking longer time with Running buffer too dilute Make fresh running buffer and use a 1X dilution.
than usual dilute from concentrate or remake if necessary. recommended voltage

Upper buffer chamber is leaking Make sure the buffer core is firmly seated, the gaskets are in Current too high and Running buffer too concentrated Make fresh running buffer and use a 1X dilution.
place and the gel tension lever is locked. excessive heat generated
with recommended voltage
Voltage is set too low Set correct voltage.
Current too low or no current Incomplete circuit Remove the tape from the bottom of the gel cassette
Current reading on power Tape left on the bottom of the cassette Remove tape from bottom of cassette. with recommended voltage prior to electrophoresis. Make sure the buffer covers
supply is zero or very low sample wells; check the wire connections on the
Connection to power supply Check all connections with a voltmeter for conductance. buffer core.
not complete
Streaking of proteins Sample overload Load less protein.
Insufficient buffer level Make sure the upper buffer (cathode) is covering the wells of
the gel. Be sure there is sufficient buffer in the Lower Buffer High salt concentration in sample Decrease the sample salt concentration by dialysis or
Chamber to cover the slot at the bottom of the gel. gel filtration.

Run is faster than normal with Buffers are too concentrated Check buffer recipe; dilute or re-make if necessary. Sample precipitates Increase the concentration of SDS in the sample.
poor resolution or incorrect
Contaminants such as lipids or DNA com- Centrifuge or clarify the sample to remove particulate
Voltage, current, or wattage is set at a Decrease power conditions to recommended running plexes in sample contaminants. Treat sample with nuclease(s).
higher limit conditions (see page 59).
Poorly poured gel Make sure the gel is poured evenly and all at once.
Cannot see the sample wells There is little contrast between the Mark cassette at the bottom of the wells with a marker pen
to load sample sample well and the rest of the gel prior to assembling the Upper Buffer Chamber. Illuminate Fuzzy bands Protein sample only partially denatured Fully denature the protein.
the bench area with a light source placed directly behind the Protein sample only partially reduced Make sure a sufficient amount of DTT or
XCell SureLock unit. -mercaptoethanol is added.

Mini Gel Tank troubleshooting Gel runs for too long Watch the dye front as an indicator for proper
running time.
Observation Cause Solution
Dumbbell shaped bands or Loading a large volume of sample causes Load appropriate volume of sample.
Run taking longer than usual Buffers are too dilute Check buffer recipe; dilute from concentrate or remake
smiling bands incomplete stacking If the sample is too dilute, concentrate it using
if necessary.
ultrafiltration.
Buffer chamber is leaking Make sure the cassette clamp is firmly seated, the gaskets
Uneven electric field during run Try to make sure the loading is symmetrical if the protein
are in place and the cassette clamp is locked.
concentration is known.
Current is set too low Set correct current.
Uneven surface of the resolving gel Try to make the resolving gel surface even while pouring
Current reading on power Tape left on the bottom of the cassette Remove tape from bottom of cassette. the gel.
supply is zero or very low
Connection to power supply Check all connections with a voltmeter for conductance. Expired gels Use the gels before the specified expiration date; Note:
not complete NuPAGE gels have an extended 12 month shelf life,
minimizing the risk of having expired gels.
Insufficient buffer level Make sure there is sufficient buffer in the electrophoresis tank
to cover the wells of the gel.

Run is faster than normal with Buffers are too concentrated Check buffer recipe; dilute or re-make if necessary.
poor resolution or incorrect

Current is set at a higher limit Decrease current to recommended running conditions (see
page 59).

Cannot see the sample wells There is little contrast between the Mark cassette at the bottom of the wells with a marker pen
to load sample sample well and the rest of the gel prior to placing the cassette in the electrophoresis tank.

Sample preparation and Electrophoresis chamber Electrophoresis


Precast protein gels Protein standards Protein gel stains
electrophoresis buffers systems and power supplies run conditions
Prepare samples Choose the electrophoresis
62 Select precast gel and select buffers Select the standard chamber system and power supply Run the gel Stain the gel Post stain Protein gel electrophoresis technical handbook 63

Stain the gel


Protein stains Silver stains Coomassie dye
Thermo Scientific Pierce Silver Stain
protein gel stains
Once protein bands have been Invitrogen SilverXpress Silver Stain

Thermo Scientific Pierce Silver Stain for Mass Spectrometry


separated by electrophoresis, Convenient, ready-to-use reagents with
Fluorescent/specialty stains no permanent chemical modification
they can be directly visualized Invitrogen SYPRO Orange, Red, or Ruby gel stain

using different methods of in-gel Pierce Reversible stain for nitrocellulose or PVDF membranes The most common methods for in-gel protein
detection use stains with Coomassie dye. These
Pro-Q Emerald Glycoprotein stain
detection. Over the past several stains use either the G-250 (colloidal) or R-250
Pro-Q Diamond Phosphoprotein stain form of the dye (Table 6). Colloidal Coomassie stain
decades, demands for improved can be formulated to effectively stain proteins within Our Coomassie stains provide sensitive protein detection along
To visualize the proteins, a protein-specific, dye-binding or one hour and require only water (no methanol or with simplified protocols. Example data and staining protocols are
sensitivity for small sample sizes color-producing chemical reaction must be performed on the acetic acid) for destaining. shown for SimplyBlue SafeStain (Figure 30, 33 and 34), PageBlue
proteins within the gel. Depending on the particular chemistry Protein Staining Solution (Figure 32), and Imperial Protein Stain
and compatibility with downstream of the stain, various steps are necessary to hold the proteins Key features: (Figure 31 and 35).

applications and detection in the matrix and to facilitate the necessary chemical reaction.
Most staining methods involve some version of the same general
SimpleCoomassie dyebased formulations are easy to
Learn more at
formulate and are widely used
instrumentation have driven the incubation steps:
Easy to usesimply soak the gel in stain solution and
thermofisher.com/coomassiestains

destain to observe protein bands


development of several basic DI H2O S DI H2O S

EconomicalCoomassie dyebased stain formulations are


staining methods. Each method cost effective
1. Water wash. 2. Fix. 3. Water wash. 4. Stain. 5. Destain.

has particular advantages and Flexibleuseful for qualitative visualization, quantitative


densitometry, and gel excision and analysis by
A water-wash to remove electrophoresis buffers from the
disadvantages, and a number gel matrix
mass spectrometry

Table 6. Coomassie dyebased protein gel stains.


of specific formulations of each An acid or alcohol wash to condition or fix the gel to limit
PageBlue Protein
diffusion of protein bands from the matrix SimplyBlue SafeStain Imperial Protein Stain
Staining Solution
type of method provide optimal Treatment with the stain reagent to allow the dye or chemical to Type G-250 R-250 G-250
diffuse into the gel and bind (or react with) the proteins
performance for various situations. Destaining to remove excess dye from the background
Limit of detection >7 ng 3 ng 5 ng

Time to stain (min) 12 60 60


gel matrix
Compatible with:
Typically these stains can be classified broadly based on the dye
PVDF membranes Yes Yes Yes
or molecule that helps visualize the protein stains: Depending on the particular staining method, two or more of Nitrocellulose membranes No No No
these functions can be accomplished with one step. For example, Reusable No No Yes (up to 3x)
Coomassie stains a dye reagent that is formulated in an acidic buffer can effectively Mass spectrometry Yes Yes Yes
Thermo Scientific PageBlue stain
fix and stain in one step. Conversely, certain functions require compatible
several steps. For example, silver staining requires both a Color Purple Purple Blue-green
SimplyBlue SafeStain
staining reagent step and a developer step to produce the colored Feature Free of methanol and acetic acid Photographs better than Free of methanol
Thermo Scientific Imperial Protein Stain reaction product. Coomassie G-250 dye and acetic acid
Advantages Rapid, sensitive completely non-hazardous Fast, ultrasensitive Cost-effective
(does not require methanol or acetic acid protein detection option for fast,
Learn more at thermofisher.com/proteinstains fixatives or destains) staining sensitive staining

Sample preparation and Electrophoresis chamber Electrophoresis


For ordering information refer to page 87. Precast protein gels Protein standards Protein gel stains
electrophoresis buffers systems and power supplies run conditions
Prepare samples Choose the electrophoresis
64 Select precast gel and select buffers Select the standard chamber system and power supply Run the gel Stain the gel Post stain Protein gel electrophoresis technical handbook 65

Protocols Example data


1 2 3 4 5 6 7 8 9 10

2
3
1

S
DI H2O DI H2O DI H2O

Figure 30. SimplyBlue SafeStain protocol.

Figure 33. Sensitive staining results with SimplyBlue SafeStain. The Figure 34. Two-dimensional electrophoresis (2DE) analysis of spinach
1. Wash the gel three 2. Add SimplyBlue 3. Wash gel with 100 mL 4. Additional water wash
following samples were separated on a NuPAGE Novex 4-12% Bis-Tris chloroplast extract; staining with SimplyBlue SafeStain. Spinach
times (5 minutes) with SafeStain (1 hour). of DI water for 1 hour. with 100 mL of DI water
ultrapure water. gel and then stained with SimplyBlue SafeStain. Lane 1: 6 g protein mix; chloroplast extract was prefractionated in the ZOOM IEF Fractionator and
(1 hour) for increased
sensitivity. Lane 2: 1 g rabbit IgG; Lane 3: 1 g reduced BSA; Lane 4: 5 g the individual fractions were then separated by 2DE using narrow pH range
E. coli lysate; Lane 5: 20 ng reduced BSA; Lane 6: 10 ng reduced BSA; ZOOM Strips and NuPAGE Novex 412% Bis-Tris ZOOM Gels. Gels were
Lane 7: 7 ng reduced BSA; Lane 8: 3 ng reduced BSA; Lane 9: 10 L Coomassie stained using SimplyBlue SafeStain.
Mark12 Unstained Standard (blend of 12 purified proteins); Lane 10: 5 L
Mark12 Unstained Standard.

1 2 3 4 5 6 7 8 9 1 2 3 4 5 6 7 8 9 1 2 3 4 5 6 7 8 9

Rabbit IgG
S
DI H2O DI H2O
BSA
Protein A
Figure 31. Imperial Protein Stain protocol. Protein G

1. Wash the gel three 2. Add Imperial Protein Stain 3. Water destain Lysozyme
times with deionized (5 minutes1 hour). (15 minutesovernight).
water (15 minutes). 5-minute stain; 1-hour stain; 1-hour stain;
15-minute water destain 2-hour water destain overnight water destain
Figure 35. Enhanced sensitivity and clear background using Imperial Protein Stain. For even
greater sensitivity and reduced background, gels can be stained with Imperial Protein Stain for 1 hour
and washed with water from 1 hour to overnight. Lane 1: BSA only (6 g); Lane 29: loaded left to right
with 1,000, 200, 100, 50, 25, 12, 6, and 3 ng protein sample.

Did you know?


Staining with a Coomassie stain
DI H2O S DI H2O DI H2O prior to silver staining allows for
Recommended products more uniform staining of certain
proteins since silver ions can
The Thermo Scientific Pierce Power Stainer is designed for rapid
interact with certain functional
Coomassie dye staining of proteins in up to two minigels and subsequent
groups such as carboxylic acid
1. Wash the gel three 2. Add PageBlue Protein 3. Rinse gel two times 4. Wash gel one time removal of unbound stain from the gel in a single step. Refer to page 72 of
Figure 32. PageBlue Protein Staining groups, imidazole, sulfhydryls,
times with ultrapure Staining Solution (1 hour). with ultrapure water with ultrapure water Solution protocol. this brochure for more information.
water (30 minutes). (<1 minute) (5 minutes) and amines.

Sample preparation and Electrophoresis chamber Electrophoresis


For ordering information refer to page 87. Precast protein gels Protein standards Protein gel stains
electrophoresis buffers systems and power supplies run conditions
Prepare samples Choose the electrophoresis
66 Select precast gel and select buffers Select the standard chamber system and power supply Run the gel Stain the gel Post stain Protein gel electrophoresis technical handbook 67

Silver stains Table 7. Silver stain kits.

Pierce Silver Stain for SilverXpress Silver


Pierce Silver Stain Kit
Mass Spectrometry Staining Kit
Ultra-sensitive stains with optimized
Components (steps) 6 (17) 4 (15) 5 (13)
protocols, manufactured for
Time required 1 hr 13 min 2 hr 25 min 2 hr
minimal variability
Limit of detection 0.25 ng 0.25 ng 0.86 ng

Silver staining is the most sensitive colorimetric Mass spectrometry Yes Yes Yes
compatible
method for detecting total protein, and functions
Storage Room temperature Room temperature 4C
by the deposition of metallic silver at the location of
protein bands. Silver ions (from silver nitrate in the Stability 1 year 1 year 6 months
stain reagent) interact and bind with certain protein Advantages Fast and sensitive staining and destaining of Rapid, ultrasensitive and Nanogram-level
functional groups. The strongest interactions protein gels versatile silver stain system sensitivity for silver
occur with carboxylic acid groups (Asp and Glu), Optimized for peptide recovery after in-gel Flexible gel fixation (30 min to staining with minimal
typsin digestion for mass spectrometry overnight) and staining (5 min to background
imidazole (His), sulfhydryl groups (Cys), and amines Flexible gel fixation (1530 min to overnight) 20 hours)
(Lys). Various sensitizer and enhancer reagents are and staining (130 min)
essential for controlling the specificity and efficiency We offer highly sensitive silver stains with short protocol times
of silver ion binding to proteins and effective that are also compatible with mass spectrometry (Table 7). The Protocols and example data
conversion (development) of the bound silver to SilverXpress Silver Staining Kit provides nanogram-level sensitivity
metallic silver. with minimal background (Figure 37), while the Pierce Silver Stain
F SZ
H2O
Kit provides protocol flexibility (Figure 38 and 39).
1 2 3 4 5 6 7 8 9 10
Key features:
Sensitivesilver stains are highly sensitive stains that allow
Learn more at thermofisher.com/silverstains
1. Wash the gel 2. Fix the gel in Fixing Solution 3. Decant the Fixing Solution
for visualization of proteins at sub-nanogram levels with water. for 10 minutes. and incubate the gel in 2
changes of Sensitizing Solution.
Easy to use and flexibleoptimized for minimal steps and
flexibility to accommodate shorter or longer protocols S
H2 O
Workflow compatibleour mild chemical formulations
help ensure compatibility with mass spectrometry S
and sequencing
4. Decant the Sensitizing Solution 5. Incubate the gel in Staining Solution.
and rinse the gel two times with Figure 37. Crystal clear background with the SilverXpress Kit. Samples
Robust performancedetailed protocol enables ultrapure water. were separated on a NuPAGE Novex 4-12% Bis-Tris gel and stained with
consistent results with clear background the SilverXpress Kit. Lanes 1, 10: Mark12 Unstained Standard (blend of 12
H2O D purified proteins) diluted 1:4; Lane 2: Mark12 Unstained Standard diluted
1:8; Lane 3: Mark12 Unstained Standard diluted 1:16; Lane 4: Mark12
Unstained Standard diluted 1:32; Lane 5: Mark12 Unstained Standard
D
diluted 1:64; Lane 6: 1.6 ng BSA; Lane 7: 0.8 ng BSA; Lane 8: E. coli
6. Decant the Staining Solution 7. Incubate the gel in Developing Solution. lysate diluted 1:20; Lane 9: E. coli lysate diluted 1:80.
and rinse the gel two times with
ultrapure water.

SS
H 2O

8. Add the Stopping Solution directly 9. Decant the Stopping Solution and wash
to the gel when the desired the gel three times with ultrapure water.
staining intensity is reached.

Figure 36. SilverXpress Silver Staining Kit protocol.

Sample preparation and Electrophoresis chamber Electrophoresis


For ordering information refer to page 87. Precast protein gels Protein standards Protein gel stains
electrophoresis buffers systems and power supplies run conditions
Prepare samples Choose the electrophoresis
68 Select precast gel and select buffers Select the standard chamber system and power supply Run the gel Stain the gel Post stain Protein gel electrophoresis technical handbook 69

EtOH
Acetic
Acid
F
Lysate staining MW marker staining
Fluorescent protein gel stains
H2O

Rapid, highly sensitive fluorescent Learn more at


F thermofisher.com/fluorescentstains
stains for total protein detection
1. Wash 2 x 5 minutes 2. Fix 2 x 15 minutes in EtOH/
with ultrapure water. acetic acid. after electrophoresis Recommended products
For optimal sensitivity with Polaroid film, SYPRO Photographic Filter
SZ Fluorescent gel stains are designed for use in 1D is recommended.
10% EtOH
and 2D PAGE and offer sensitivities similar to that
obtained with silver staining techniques. Invitrogen
SZ
SYPRO protein stains are easy-to-use fluorescent
3. Incubate 2 x 5 minutes 4. Mix Sensitizer. Sensitize for 2 minutes, 30 seconds 2 minutes, 30 seconds
with 10% EtOH. Wash. 1 minute. Wash 2 x 1 minute. stains for the detection of proteins separated by
Development Time
PAGE (Table 8). Stained proteins can be viewed
Figure 39. Pierce Silver Stain Kit exhibits excellent senstivity. In with a standard UV or blue-light transilluminator or
standard minigels, proteins are detectable at greater than 0.25 ng per
S with a laser scanner.
band or spot.

S Features:

5. Mix Silver Stain. Stain for 5 minutes. Simpleno destaining or timed steps required; minimal
Wash 2 x 20 seconds. hands-on time

Quantitativelinear quantitation range over two orders of


D
5% Acetic Acid magnitude with low protein-to-protein variability

Highly sensitivetypically more sensitive than Coomassie


D dyebased stains and equivalent to silver stains

6. Mix Developer. Develop for 2-3 minutes. 7. Remove developer.


Stop with 5% Acetic
Acid for 10 minutes.
Table 8. SYPRO protein stains.

Figure 38. Pierce Silver Stain Kit protocol. SYPRO Ruby stain SYPRO Orange stain SYPRO Red stain

Limit of detection 0.25 ng 48 ng 48 ng

Stain and destain time 90 min microwave; 18 hr standard ~1 hr ~1 hr

Ex/Em 280 nm, 450/610 nm 300 nm, 470/510 nm 300 nm, 550/630 nm

Ease of use Ready to use Supplied as stock solution Supplied as stock solution

Compatible applications Mass spectrometry, IEF, 2D gels, Mass spectrometry, IEF, 2D gels, Mass spectrometry, IEF, 2D gels,
on-membrane staining on-membrane staining on-membrane staining

Sample preparation and Electrophoresis chamber Electrophoresis


For ordering information refer to page 87. Precast protein gels Protein standards Protein gel stains
electrophoresis buffers systems and power supplies run conditions
Prepare samples Choose the electrophoresis
70 Select precast gel and select buffers Select the standard chamber system and power supply Run the gel Stain the gel Post stain Protein gel electrophoresis technical handbook 71

Stain the gel


Specialty protein stains Electrophoretic staining technologyPierce Power Stainer
Our specialty protein stains include in-gel Learn more at thermofisher.com/specialtystains The Thermo Scientific Pierce
phosphoprotein and glycoprotein detection and Good to know
on-membrane reversible protein staining kits (Table 9). Power Stainer consists of a
How does electrostaining work?
Table 9. Specialty protein stains. Thermo Scientific Pierce Power Cathode ()

Pro-Q Emerald 488 Glycoprotein


Gel and Blot Stain Kit
Pro-Q Emerald 300 Glycoprotein Gel
and Blot Stain Kit
Pro-Q Diamond Phosphoprotein Gel
Staining Kit
Station with activated Staining Coomassie staining pad

Software and a Thermo Scientific


Pre-run and pre-washed SDS-PAGE gel
Detects Glycoproteins Glycoproteins Phosphoproteins
Destaining pad

Sensitivity 4 ng glycoprotein per band 0.5 ng glycoprotein per band 116 ng phosphoprotein per band Pierce Power Stain Cassette. It Anode (+)

Stain and ~6 hr ~5 hr 45 hr
is designed for rapid Coomassie
destain time
staining and destaining of proteins Pierce Power Stain Cassette

Ex/Em 510/520 nm 280/530 nm 555/580 nm


in polyacrylamide gels. Traditional Cathode ()

Staining pad
Advantages Selective staining of glycoproteins Selective staining of glycoproteins Selective staining of phosphoproteins
Coomassie staining techniques Gel

Destaining pad

require one hour to overnight Anode (+)

staining and destaining to achieve The significant reduction in protein staining time is
accomplished by utilizing inonic Power Stain Solution and
desired results. When used in Destain Solution to electrophoretically drive the negatively

conjunction with Thermo Scientific charged Coomassie R-250 dye out of the top gel pad, through
the polyacrylamide gel matrix and the bottom gel pad, and

Pierce Midi and Mini Gel Power toward the positively charged anode.

Staining Kits, the Pierce Power


Stainer is designed to provide
staining efficiency in as few as
6 minutes that is equivalent to, or
better than, traditional Coomassie
staining techniques. Watch our Pierce Power Stainer video.
thermofisher.com/powerstainer

Sample preparation and Electrophoresis chamber Electrophoresis


For ordering information refer to page 87. Precast protein gels Protein standards Protein gel stains
electrophoresis buffers systems and power supplies run conditions
Prepare samples Choose the electrophoresis
72 Select precast gel and select buffers Select the standard chamber system and power supply Run the gel Stain the gel Post stain Protein gel electrophoresis technical handbook 73

Pierce Power Stainer


Rapid Coomassie dye staining and
destaining in approximately 10 minutes

The Thermo Scientific Pierce Power Stainer


is designed for rapid Coomassie dye staining of
proteins in polyacrylamide gels and subsequent
removal of unbound stains to give sharply stained
protein bands with minimal or no background.

The Pierce Power Stainer offers: Specifications


SpeedCoomassie dye staining and destaining of proteins Mode of transfer: semi-dry blotting
in about 10 minutes Recommended products
Gel compatibility: SDS-PAGE gels
The Pierce Power System can be used both for fast Coomassie dye
Conveniencesimultaneously stain and destain 12
Running dimension: horizontal staining of protein gels and for rapid semi-dry transfer of proteins from gel
minigels or 1 midigel to membrane. The Pierce Power Stainer can be upgraded by adding the
Platform: Pierce Power System Pierce Power Blot Cassette to make a fully functional Pierce Power
Reliable performanceenables staining results that are System with blotting and staining capabilities.
equivalent to traditional staining techniques

Easy touch programmingintuitive LCD touch-screen


Pierce Power Stainer Conventional Coomassie stain
interface includes preprogrammed protocols
Did you know?
Conventional Coomassie
dyebased staining techniques
require 1 hour to overnight
incubation.

Total time: 11 minutes Total time: 230 minutes


to overnight
1. Wash gel 1 5 minutes in 1. Wash gel 3 10 minutes in water Thermo Scientific Pierce Thermo Scientific Pierce
water 2. Incubate gel in Coomassie Power Stainer Power Blotter
2. Power Stain/Destain gel, 6 stain solution* for 60 minutes
minutes 3. Wash gel 2 10 minutes in water
4. Destain gel in destaining solution** for
3 20 minutes
5. Incubate gel in water for 60 minutes
to overnight

*Coomassie stain solution: 45% methanol, 10% acetic acid, 0.25% Coomassie R-250
**Destain solution: 30% ethanol, 5% acetic acid

Figure 40. Pierce Power Stainer saves time and maintains sensitivity.

Learn more at thermofisher.com/powerstainer

Sample preparation and Electrophoresis chamber Electrophoresis


For ordering information refer to page 87. Precast protein gels Protein standards Protein gel stains
electrophoresis buffers systems and power supplies run conditions
Prepare samples Choose the electrophoresis
74 Select precast gel and select buffers Select the standard chamber system and power supply Run the gel Stain the gel Post stain 75 75
Protein gel electrophoresis technical handbook

Western blotting
Transfer and Detection
After electrophoresis, the Horseradish peroxidase (HRP) or alkaline phosphatase Key products for western blot transfer:
(AP) are the most popular enzymes conjugated to
separated proteins are transferred antibodies used in the western blot workflow. After Wet Semi-dry Dry

incubating the membrane with the detection antibody


or blotted onto a second matrix, or antibodies, if an enzyme-conjugated antibody
was utilized, an appropriate substrate (chromogenic
generally a nitrocellulose or or chemiluminescent) is added and that results in a
detectable product. A popular substrate of choice is
polyvinylidene difluoride (PVDF) a chemiluminescent substrate that, when combined
with the enzyme, produces light as a byproduct. With
membrane. Next, the membrane the chemiluminescent substrate, the light output can
be captured on film or CCD camera. In recent years
is blocked to minimize potential fluorescent detection became a popular alternative
Mini Blot Module Thermo Scientific Pierce
Power Blotter
iBlot 2 Dry Blotting System

nonspecific binding of antibodies to the enzymatic detection since it allows for more
quantitative data analysis. Fluorescent detection
to the surface of the membrane. utilizes dye-labeled primary antibodies or dye- Key products for western blot detection include:
labeled secondary antibodies and the signal output is
captured on an appropriate imaging system. Whatever Automated detection Manual detection
Detailed procedures vary widely for the detection substrate is used, the intensity of the signal should
steps of the western blot workflow. One common correlate with the abundance of the antigen on the
Blocking buffers
variation involves direct vs. indirect detection methods. Wash buffers
blotting membrane. Detergents
In both the direct and indirect detection methods, Enhancers
the blocked membrane is probed with an antibody We offer a wide range of reagents, kits, equipment, Substrates
(primary antibody) specific to the protein of interest and antibodies to facilitate every step of western Stripping buffers
X-ray film
(antigen). In direct detection techniques, this antibody blot analysis.
is enzyme conjugated or labeled with a fluorophore.
However, in indirect detection techniques, the blocked
iBind Flex Western Device
membrane is probed first with an antibody (primary
antibody) which is specific to the antigen followed
by another antibody (secondary antibody) raised
against the host species of the primary antibody. This
secondary antibody is often enzyme conjugated or
labeled with a fluorophore. The direct method is not
widely used as most researchers prefer the indirect
detection method for a variety of reasons.

Learn more at thermofisher.com/western

Sample preparation and Electrophoresis chamber Electrophoresis


Precast protein gels Protein standards Protein gel stains
electrophoresis buffers systems and power supplies run conditions
76 Appendix Protein gel electrophoresis technical handbook 77

Protocol quick reference


QUICK REFERENCE QUICK REFERENCE

NuPAGE Bis-Tris Midi Gels NuPAGE Tris-Acetate Midi Gels


Bolt Mini Gels Bolt Mini Gels
Instructions for electrophoresis of Bis-Tris Gels using the XCell4 SureLock Midi-Cell are
Instructions for performing electrophoresis using Bolt Mini Gels are described below. described below. Prepare Reagent Denaturing Native Sample
Prepare gel 1. Cut open the gel cassette pouch and remove the cassette. Samples Sample
and tank 2. Remove the gel comb and rinse wells 3 times with 1X Running Buffer. Sample x L x L
Prepare Reagent Reduced sample Non-reduced
Prepare Reagent Reduced Sample Non-reduced Sample 3. Remove the tape covering the slot at the lower portion of the cassette. NuPAGE LDS Sample Buffer (4X) 2.5 L
Samples sample
samples Sample x L x L Sample x L x L Tris-Glycine Native Sample Buffer (2X) 5 L
Load 1. Pre-fill the chamber with 1X Running Buffer to the level of the cathode. NuPAGE Reducing Agent (10X)* 1 L
Bolt LDS Sample Buffer (4X) 10 L 10 L NuPAGE LDS Sample Buffer (4X) 2.5 L 2.5 L
Bolt Reducing Agent (10X) 4 L samples 2. Place the cassette in the chamber with the wells facing towards you. Deionized Water to 10 L final to 10 L final
NuPAGE Reducing Agent (10X) 1 L
Deionized Water to 26 L to 30 L Hold the cassette in a raised position and close the cassette clamp.
Deionized Water to 10 L final to 10 L final *For reduced samples only.
Total Volume 40 L* 40 L* 3. Fill all wells with 1X Running Buffer.
Heat samples at 70C for 10 minutes. Heat denaturing samples at 70C for 10 minutes. Do not heat native samples.
Heat samples at 70C for 10 minutes. 4. Load your samples and markers.
* Scale samples up or down by adjusting all volumes proportionally. 5. Hold the cassette and release the cassette clamp. Prepare Add 50 mL 20X NuPAGE MES or MOPS SDS Running Buffer to 950 mL Prepare Denaturing samples: Add 50 mL 20X NuPAGE Tris-Acetate SDS Running Buffer
1X Buffer deionized water to prepare 1X SDS Running Buffer. 1X Buffer to 950 mL deionized water. Native samples: Add 100 mL 10X Tris-Glycine
6. Gently lower the cassette to the bottom of the chamber, and close the Native Running Buffer to 900 mL deionized water.
Prepare 1X Each chamber of the tank requires 400 mL of 1X SDS Running Buffer (mix cassette clamp Load Sample Load the appropriate concentration of your protein sample on the gel.
Buffer 20 mL of 20X Bolt MES or MOPS SDS Running Buffer with 380 mL of
7. Add 1X buffer to the level of the fill line. Load Sample Load the appropriate concentration of your protein sample on the gel.
deionized water). The same buffer type must be used for both chambers.
Add Buffer Fill Upper Buffer Chamber with 175 mL 1X NuPAGE SDS Running Buffer.
14 57 For reduced samples, use 175 mL 1X NuPAGE SDS Running Buffer with Add Buffer Fill Upper Buffer Chamber with 175 mL of the appropriate 1X Running Buffer.
Run Run Bolt Mini Gels at constant voltage (1 or 2 mini gels). Fill Line
435 L NuPAGE Antioxidant in the Upper Buffer Chamber. Add a sufficient For reduced samples, use 175 mL 1X Running Buffer with 435 L NuPAGE
conditions volume of 1X NuPAGE SDS Running Buffer to the Lower Buffer Chamber. Antioxidant in the Upper Buffer Chamber. Add a sufficient volume of Running
Running Buffer Standard Run Run Time*
Buffer the Lower Buffer Chamber.
MES 200 V 22 min
Run Voltage: 200 V constant
Conditions Run Time: 40 min (MES Buffer), 55 min (MOPS Buffer) Run Voltage: 150 V constant
MOPS 200 V 32 min Cathode Cathode
Expected Current: 160200 mA/gel (start); 120170 mA/gel (end) Conditions Run Time: 70 min (denaturing gel), 23 hours (native gel)
* Run times may vary depending upon gel type and power supply. Expected Current: 7090 mA/gel (start); 5060 mA/gel (end); (denaturing gel)
4045 mA/gel (start); 1520 mA/gel (end); (native gel)
For research use only. Not for use in diagnostic procedures. For Research Use Only. Not for use in diagnostic procedures.

QUICK REFERENCE
QUICK REFERENCE

NuPAGE Bis-Tris Mini Gels NuPAGE Tris-Acetate Mini Gels Novex Tris-Glycine Mini Gels Novex Tris-Glycine Mini Gels
Instructions for electrophoresis using the XCell SureLock Mini-Cell are described below. Instructions are provided below for electrophoresis of NovexTris-Glycine Gels using the Non-Denaturing (Native) Electrophoresis
Prepare Reagent Denatur i ng Sampl e* Nati ve Sampl e
XCellSureLock Mini-Cell.
Samples Sam pl e x L x L
Prepare Reagent Reduced Sampl e Non- r educed Sampl e Prepare Reagent Sampl e
NuPAGE LDS Sample Buffer (4X) 2.5 L -- Denaturing Electrophoresis
Samples Sam pl e x L x L Tris-Glycine Native Sample Buffer (2X) -- 5 L Samples Sample x L
NuPAGE LDS Sample Buffer (4X) 2.5 L 2.5 L D ei on i z ed Water t o 7.5 L to 5 L Tris-Glycine Native Sample Buffer (2X) 5 L
Prepare Reagent Reduced Sample Non-reduced Sample
Deionized Water to 5 L
NuPAGE Reducing Agent (10X) 1 L -- Tot al Vol u m e 10 L 10 L Samples Sample x L x L Total Volume 10 L
Deionized Water to 6.5 L to 7.5 L Samples Heat samples at 70C for 10 minutes Do not heat Tris-Glycine SDS Sample Buffer (2X) 5 L 5 L
Do not heat samples for native electrophoresis.
Tot al Vol u m e 10 L 10 L *For reduced samples, add NuPAGE Reducing Agent (10X) to 1X. NuPAGE Reducing Agent (10X) 1 L --
Deionized Water to 4 L to 5 L
Heat samples at 70C for 10 minutes. Prepare 1X Add 100 mL 10X Tris-Glycine Native Running Buffer to 900 mL deionized
Prepare 1X Denaturing Samples: Add 50 mL 20X NuPAGE Tris-Acetate SDS Running Total Volume 10 L 10 L water to prepare 1X Tris-Glycine Native Running Buffer.
Buffer
Buffer Buffer to 950 mL deionized water. Native Samples: Add 100 mL 10X Tris-
Prepare 1X Add 50 mL 20X NuPAGE MES or MOPS SDS Running Buffer to 950 mL
Glycine Native Running Buffer to 900 mL deionized water. Heat samples at 85C for 2 minutes.
Buffer deionized water to prepare 1X SDS Running Buffer. Load Sample Load the appropriate concentration of your protein sample on the gel.
Prepare 1X Add 100 mL 10X Novex Tris-Glycine SDS Running Buffer to 900 mL de-
Load Sample Load the appropriate concentration of your protein sample on the gel. Buffer
Load Sample Load the appropriate concentration of your protein sample on the gel. ionized water to prepare 1X Tris-Glycine SDS Running Buffer. Load Buffer Fill the Upper Buffer Chamber with 200 mL and the Lower Buffer Chamber
with 600 mL of 1X Tris-Glycine Native Running Buffer.
Load Buffer Fill the Upper (200 mL) and Lower (600 mL) Buffer Chambers with the
Load Buffer Fill the Upper (200 mL) and Lower (600 mL) Buffer Chambers with the ap- appropriate 1X Running Buffer. For reduced samples, use 200 mL 1X Load Sample Load the appropriate concentration of your protein sample on the gel.
propriate 1X Running Buffer. For reduced samples, use 200 mL 1X Running Run Voltage: 125 V constant
Running Buffer with 500 L NuPAGE Antioxidant in the Upper Buffer Chamber.
Buffer with 500 L NuPAGE Antioxidant in the Upper Buffer Chamber. Load Buffer Run Time: 112 hours
Fill the Upper Buffer Chamber with 200 mL and the Lower Buffer Chamber Conditions
Run Voltage: 150 V constant with 600 mL of 1X Tris-Glycine SDS Running Buffer. Expected Current: 612 mA/gel (start); 36 mA/gel (end)
Run Voltage: 200 V constant Run Time: 1 hour (Denaturing gel), 23 hours (Native gel)
Conditions
Conditions Run Time: 35 minutes (MES Buffer), 50 minutes (MOPS Buffer) Expected 4055 mA/gel (start); 2540 mA/gel (end) for denaturing gel Run Voltage: 125 V constant Blot Gel For blotting denaturing and native gels, use 1X Tris-Glycine Transfer Buffer
Expected Current: 100125 mA/gel (start); 6080 mA/gel (end) Current: 18 mA/gel (start); 7 mA/gel (end) for native gel Run Time: 90 minutes (dependent on gel percentage) with 20% methanol. Perform blotting at 25 V constant for 12 hours using
Conditions the XCell II Blot Module. The expected start current is 100 mA.
Expected Current: 3040 mA/gel (start); 812 mA/gel (end)
Intended Use: For research use only. Not for human or animal therapeutic or diagnostic use.
Intended Use: For research use only. Not for human or animal therapeutic
or diagnostic use.

Appendix
78 Appendix Protein gel electrophoresis technical handbook 79

Protocol quick reference


QUICK REFERENCE QUICK REFERENCE

Tris-Glycine Midi Gels Non-denaturing (Native) Electrophoresis Tricine Gels Tricine Gels
Instructions for electrophoresis using the XCell4 SureLock Midi-Cell are described below. Blotting
Prepare Reagent Native Sample Instructions are provided below for electrophoresis of Tricine Gels using the XCell SureLock For blotting Tricine gels, use 1X Tris-Glycine Transfer Buffer with
Mini-Cell. Conditions 20% methanol. Perform transfer with nitrocellulose or PVDF membranes at
Prepare Samples Sample x L
Reagent Reduced sample Non-reduced 25 V constant for 12 hours using the XCell II Blot Module. The expected
Samples sample Tris-Glycine Native Sample Buffer (2X) 5 L start current is 100 mA.
Sample x L x L Deionized Water to 10 L final Prepare Reagent Reduced Sampl e Non- r educed Sampl e
Tris-Glycine SDS Sample Buffer (2X) 5 L 5 L Do not heat native samples. Samples Alternate The Tris-Glycine Transfer Buffer interferes with protein sequencing. If you
Sam pl e x L x L Transfer
NuPAGE Reducing Agent (10X) 1 L are performing protein sequencing, use 1X NuPAGE Transfer Buffer or
Tricine SDS Sample Buffer (2X) 5 L 5 L Buffers 0.5X TBE Transfer Buffer for blotting.
Prepare Add 100 mL 10X Tris-Glycine SDS Running Buffer to 900 mL deionized water to
Deionized Water to 10 L final to 10 L final NuPAGE Reducing Agent (10X) 1 L --
1X Buffer prepare 1X Tris-Glycine SDS Running Buffer. The NuPAGE Transfer Buffer protects against modification of the amino
Heat samples at 85C for 2 minutes. Deionized Water to 4 L to 5 L acid side chains and is compatible with N-terminal protein sequencing
Load Sample Load the appropriate concentration of your protein sample on the gel. Tot al Vol u m e 10 L 10 L using Edman degradation.
Prepare Add 100 mL 10X Tris-Glycine SDS Running Buffer to 900 mL deionized water to
Heat samples at 85C for 2 minutes.
1X Buffer prepare 1X Tris-Glycine SDS Running Buffer. Add Buffer Fill each Upper Buffer Chamber with 175 mL of 1X Tris-Glycine Native Running
Buffer. Fill the Lower Buffer Chamber up to the fill line mark with 1X Tris-
Load Sample Load the appropriate concentration of your protein sample on the gel. Glycine Native Running Buffer.
Prepare 1X Add 100 mL 10X Novex Tricine SDS Running Buffer to 900 mL deionized
Buffer water to prepare 1X Tricine SDS Running Buffer.
Add Buffer Fill each Upper Buffer Chamber with 175 mL 1X Tris-Glycine SDS Running Buffer. Run Voltage: 125 V constant
Fill the Lower Buffer Chamber up to the fill line mark with 1X Tris-Glycine Conditions Run Time: 112 hours Load Sample Load the appropriate concentration of your protein sample on the gel.
SDS Running Buffer. Expected Current: 3540 mA/gel (start); 1520 mA/gel
Voltage: 125 V constant Load Buffer Fill the Upper Buffer Chamber with 200 mL and the Lower Buffer Chamber
Run with 600 mL of 1X Tricine SDS Running Buffer.
Run Time: 105 min (dependent on gel percentage)
Conditions Expected Current: 4050 mA/gel (start); 2025 mA/gel (end) Run Voltage: 125 V constant
Conditions Run Time: 90 minutes (dependent on gel percentage)
For Research Use Only. Not for use in diagnostic procedures. Expected Current: 80 mA/gel (start); 40 mA/gel (end)

For research use only. Not for human or animal therapeutic or diagnostic use.

QUICK REFERENCE QUICK REFERENCE

NativePAGE Bis-Tris Gels



Staining Protocol IEF Gels IEF Gels
A quick staining protocol for NativePAGE Gels using the Coomassie G-250 from the sample
Instructions are provided below for electrophoresis of NativePAGE Bis-Tris Gels using the XCell additive is described below. The total staining time is ~23 hours. Sensitivity is ~60 ng BSA. Instructions are provided below for electrophoresis of IEF Gels using the XCell SureLock Prepare for 1. Stain and destain the IEF gel. Incubate the IEF gel in 100 mL 20% ethanol
SureLock Mini-Cell. Mini-Cell. 2D SDS/ for 10 minutes.
Step Action Time
PAGE 2. Cut out the desired lane (strip) from the gel for transfer to a SDS gel.
Place the gel in 100 mL fixing solution (40% methanol, 10% acetic 45
Prepare Reagent Sample with Detergent-free 1 Prepare Reagent Sample 3. Incubate the gel strip in 2 mL 2X SDS sample buffer and 0.5 mL ethanol for
acid) and microwave on high (9501100 watts). seconds
Samples detergent sample Samples Sample x L 35 minutes. Aspirate the sample buffer and rinse the gel strip with 1X SDS
Sample x L x L
Shake the gel on an orbital shaker. 15 Running Buffer.
2 IEF Sample Buffer pH 310 or pH 37 (2X) 5 L
minutes
NativePAGE Sample Buffer (4X) 2.5 L 2.5 L Deionized Water to 10 L final 4. Fill the SDS gel cassette with 1X SDS Running Buffer.
NativePAGE 5% G-250 Additive 0.251 L* 3 Discard fixing solution. 5. Trim the IEF gel strip to a length of 5.85.9 cm.
Deionized Water to 10 L to 10 L Prepare 1X IEF Anode Buffer: Add 20 mL 50X IEF Anode Buffer to 980mL deion-
Place the gel in 100 mL destain solution (8% acetic acid) and micro- 45 6a. Transfer the gel strip into a 1.0 mm SDS gel by sliding the strip into the
4 1X Buffer ized water. Chill to 4C to 10C.
Do not heat samples for native gel electrophoresis. wave on high (9501100 watts). seconds 2D-well using a gel loading tip. Avoid trapping air-bubbles between the strip
1X IEF Cathode Buffer: Add 20mL IEF Cathode Buffer pH 310 (10X) or pH and the SDS gel. Wet a piece of thick filter paper (5.8 cm 4 cm) in SDS
*Ensure the final G-250 concentration is th the detergent concentration. Shake the gel on an orbital shaker until the desired background is 37 (10X) to 180 mL deionized water. Chill to 4C to 10C.
5 Running Buffer and insert the long edge of the paper into the 2D-well to
obtained.
push the gel strip into contact with the SDS gel.
Prepare 1X 1X NativePAGE Anode Buffer: Add 50 mL 20X NativePAGE Running Buffer
Load Sample Load the appropriate concentration and volume of your protein on the gel.
6b. If transferring the gel strip into a 1.5 mm SDS gel, wet 2 pieces of thin filter
Running to 950 mL deionized water. paper (5.8 cm 4 cm) in 1X SDS Running Buffer. Sandwich the gel strip
Add Buffer Fill the Upper Buffer Chamber with chilled 200 mL 1X IEF Cathode Buffer
Buffer 1X NativePAGE Cathode Buffer: Add 50 mL 20X NativePAGE Running Buf- and the Lower Buffer Chamber with chilled 600 mL 1X IEF Anode Buffer. between the two filter papers along the long edge with the edge of the strip
fer and 50 mL 20X NativePAGE Cathode Additive to 900 mL deionized water. protruding ~0.5 mm beyond the paper. Insert the sandwich into the 2D-well
Run Voltage: 100 V constant for 1 hour of the SDS gel without trapping air bubbles and push the strip down so it is
Load Sample Fill wells with 1X NativePAGE Cathode Buffer and load samples prior to filling 200 V constant for 1 hour in contact with the SDS gel.
Conditions
the cathode chamber. Load an appropriate amount of your sample on the gel. 500 V constant for 30 minutes 7. Insert gel into the mini-cell, fill the buffer chambers with 1X SDS Running
Expected Current: 7 mA/gel (start); 5 mA/gel (end) Buffer, and perform SDS-PAGE.
Add Buffer Fill the Upper Buffer Chamber with ~200 mL 1X NativePAGE Cathode Buffer. 8. After the dye front has moved into the stacking gel (~10 minutes),
Fill the Lower Buffer Chamber with ~550 mL 1X NativePAGE Anode Buffer. Stain Gel Fix the IEF gel in 12% TCA or 12% TCA containing 3.5% sulfosalicylic acid disconnect power, remove the paper, and resume electrophoresis.
for 30 minutes. Stain the IEF gel with method of choice.
Run Voltage: 150 V constant
Conditions Run Time: 90115 min (312% gel); 105120 min (416% gel)
Expected Current: 1216 mA/gel (start); 24 mA/gel (end) For Research Use Only. Not for use in diagnostic procedures.
For Research Use Only. Not for use in diagnostic procedures.

Appendix
80 Appendix Protein gel electrophoresis technical handbook 81

Ordering information
QUICK REFERENCE
Product Quantity Cat. No. Product Quantity Cat. No.
Zymogram Gels Zymogram Gels
Select precast gel Bolt Bis-Tris Plus Gels NuPAGE Novex 10% Bis-Tris Protein 1 box NP0307BOX
Instructions are provided below for electrophoresis of Zymogram Gels using the XCell Develop Gel 1. Dilute Zymogram Renaturing Buffer (10X) and Zymogram Developing Buffer
SureLock Mini-Cell. (10X) 1:9 with deionized water. Prepare 100mL of each 1X buffer per 12 Bolt 8% Bis-Tris Plus Gels, 10-well

1 box NW00080BOX Gels, 1.0 mm, 9-well
mini-gels.
Prepare 2. After electrophoresis, incubate the gel in 1X Zymogram Renaturing Buffer Bolt 8% Bis-Tris Plus Gels, 12-well 1 box NW00082BOX NuPAGE Novex 10% Bis-Tris Protein 1 box NP0315BOX
Reagent Sample
Samples for 30 minutes at room temperature with gentle agitation.
Sample x L Gels, 1.5 mm, 10-well
Tris-Glycine SDS Sample Buffer (2X) 5 L 3. Decant Zymogram Renaturing Buffer and add 1X Zymogram Developing Bolt 8% Bis-Tris Plus Gels, 15-well 1 box NW00085BOX
Buffer to the gel. NuPAGE Novex 10% Bis-Tris Protein 1 box NP0316BOX
Deionized Water to 10 L final
Do not heat or reduce samples for Zymogram gels.
4. Equilibrate the gel for 30 minutes at room temperature with gentle agitation. Bolt 8% Bis-Tris Plus Gels, 17-well 1 box NW00087BOX Gels, 1.5 mm, 15-well
5. Decant buffer and add fresh 1X Zymogram Developing Buffer to the gel.
Prepare 1X Tris-Glycine SDS Running Buffer: Add 100 mL 10X Tris-Glycine SDS 6. Incubate the gel at 37C for at least 4 hours or overnight for maximum Bolt 10% Bis-Tris Plus Gels, 10-well 1 box NW00100BOX NuPAGE Novex 12% Bis-Tris Protein 1 box NP0344BOX
1X Buffer Running Buffer to 900 mL deionized water. sensitivity. Optimize results empirically by varying the sample load or
incubation time. Bolt 10% Bis-Tris Plus Gels, 12-well

1 box NW00102BOX Gels, 1.0 mm, 1-well
Load Sample Load the appropriate concentration and volume of your protein on the gel.
Stain Gel Zymogram (Blue Casein) 416% gels do not require staining. For non-pre- NuPAGE Novex 12% Bis-Tris Protein 10 gels NP0341BOX
Add Buffer Fill the Upper Buffer Chamber with 200 mL, and the Lower Buffer Chamber stained Zymogram gels, stain the gels with Colloidal Blue Staining Kit or
Bolt 10% Bis-Tris Plus Gels, 15-well 1 box NW00105BOX
with 600 mL of 1X Tris-Glycine SDS Running Buffer. the SimplyBlue Safestain. Areas of protease activity appear as clear bands Gels, 1.0 mm, 10-well
against a dark background. Bolt 10% Bis-Tris Plus Gels, 17-well

1 box NW00107BOX
Run Voltage: 125 V constant NuPAGE Novex 12% Bis-Tris Protein 2 gels NP0341PK2
Conditions
Run Time: 90 minutes (dependent on gel percentage) Sensitivity 10% Zymogram (Gelatin) Gel: 10-6 units of collagenase Bolt 12% Bis-Tris Plus Gels, 10-well

1 box NW00120BOX
Expected Current: 3040 mA/gel (start); 812 mA/gel (end) Level 12% Zymogram (Casein) Gel: 7 10-4 units of trypsin Gels, 1.0 mm, 10-well
416% Zymogram (Blue Casein) Gel: 1.5 10-3 units of trypsin Bolt 12% Bis-Tris Plus Gels, 12-well 1 box NW00122BOX
NuPAGE Novex 12% Bis-Tris Protein 10 gels NP0342BOX
For Research Use Only. Not for use in diagnostic procedures.
Bolt 12% Bis-Tris Plus Gels, 15-well

1 box NW00125BOX Gels, 1.0 mm, 12-well
Bolt 12% Bis-Tris Plus Gels, 17-well 1 box NW00127BOX NuPAGE Novex 12% Bis-Tris Protein 2 gels NP0342PK2
Gels, 1.0 mm, 12-well
Bolt 412% Bis-Tris Plus Gels, 10-well

1 box NW04120BOX
NuPAGE Novex 12% Bis-Tris Protein 1 box NP0343BOX
Bolt 412% Bis-Tris Plus Gels, 12-well

1 box NW04122BOX
Gels, 1.0 mm, 15-well
Bolt 412% Bis-Tris Plus Gels, 15-well 1 box NW04125BOX
NuPAGE Novex 12% Bis-Tris Protein 1 box NP0349BOX
Bolt 412% Bis-Tris Plus Gels, 17-well

1 box NW04127BOX Gels, 1.0 mm, 17-well
Bolt Empty Mini Gel Cassettes 20 cassettes NW2010 NuPAGE Novex 412% Bis-Tris 1 box NP0324BOX
Bolt Empty Mini Gel Cassette Combs,

20 combs NW3010 Protein Gels, 1.0 mm, 1-well
10-well NuPAGE Novex 412% Bis-Tris 10 gels NP0321BOX
Bolt Empty Mini Gel Cassette Combs, 20 combs NW3012 Protein Gels, 1.0 mm, 10-well
12-well NuPAGE Novex 412% Bis-Tris 2 gels NP0321PK2
Bolt Welcome Pack B, 412%, 15-well

1 kit** NW0412B Protein Gels, 1.0 mm, 10-well

Bolt Welcome Pack A, 412%, 10-well 1 kit** NW0412A NuPAGE Novex 412% Bis-Tris 10 gels NP0322BOX
Protein Gels, 1.0 mm, 12-well
One box contains 10 gels.
** B
 olt Welcome Pack kit includes Mini Gel Tank, 2 boxes of Bolt 412% Gels NuPAGE Novex 412% Bis-Tris 2 gels NP0322PK2
(10-well/15-well), Bolt MES Running Buffer (20X), Bolt LDS Sample Buffer (4X), Bolt Protein Gels, 1.0 mm, 12-well
Sample Reducing Agent (10X) and SeeBlue Plus2 Prestained Standard
NuPAGE Novex 412% Bis-Tris 10 gels NP0323BOX
NuPAGE Bis-Tris Mini Gels (8 cm x 8 cm) Protein Gels, 1.0 mm, 15-well

NuPAGE Novex 10% Bis-Tris Protein 1 box NP0304BOX NuPAGE Novex 412% Bis-Tris 2 gels NP0323PK2
Gels, 1.0 mm, 1-well Protein Gels, 1.0 mm, 15-well

NuPAGE Novex 10% Bis-Tris Protein 10 gels NP0301BOX NuPAGE Novex 412% Bis-Tris 10 gels NP0329BOX
Gels, 1.0 mm, 10-well Protein Gels, 1.0 mm, 17-well

NuPAGE Novex 10% Bis-Tris Protein 2 gels NP0301PK2 NuPAGE Novex 412% Bis-Tris 2 gels NP0329PK2
Gels, 1.0 mm, 10-well Protein Gels, 1.0 mm, 17-well

NuPAGE Novex 10% Bis-Tris Protein 10 gels NP0302BOX NuPAGE Novex 412% Bis-Tris 1 box NP0327BOX
Gels, 1.0 mm, 12-well Protein Gels, 1.0 mm, 9-well

NuPAGE Novex 10% Bis-Tris Protein 2 gels NP0302PK2 NuPAGE Novex 412% Bis-Tris 10 gels NP0335BOX
Gels, 1.0 mm, 12-well Protein Gels, 1.5 mm, 10-well

NuPAGE Novex 10% Bis-Tris Protein 1 box NP0303BOX NuPAGE Novex 412% Bis-Tris 2 gels NP0335PK2
Gels, 1.0 mm, 15-well Protein Gels, 1.5 mm, 10-well

Appendix
82 Appendix Protein gel electrophoresis technical handbook 83

Ordering information
Product Quantity Cat. No. Product Quantity Cat. No. Product Quantity Cat. No. Product Quantity Cat. No.

NuPAGE Novex 412% Bis-Tris 10 gels NP0336BOX NuPAGE Novex 38% Tris-Acetate 2 gels EA0375PK2 Novex 10% Tris-Glycine Mini Protein 1 box EC6078BOX Novex 18% Tris-Glycine Mini Protein 1 box EC6505BOX
Protein Gels, 1.5 mm, 15-well Protein Gels, 1.0 mm, 10-well Gels, 1.5 mm, 10-well Gels, 1.0 mm, 10-well
NuPAGE Novex 412% Bis-Tris 2 gels NP0336PK2 NuPAGE Novex 38% Tris-Acetate 10 gels EA03752BOX Novex 10% Tris-Glycine Mini Protein 1 box EC60785BOX Novex 18% Tris-Glycine Mini Protein 1 box EC65052BOX
Protein Gels, 1.5 mm, 15-well Protein Gels, 1.0 mm, 12-well Gels, 1.5 mm, 15-well Gels, 1.0 mm, 12-well
NuPAGE Bis-Tris Midi Gels (8 cm x 13 cm) NuPAGE Novex 38% Tris-Acetate 2 gels EA03752PK2 Novex 1020% Tris-Glycine Mini 1 box EC6135BOX Novex 18% Tris-Glycine Mini Protein 1 box EC65055BOX
NuPAGE Novex 10% Bis-Tris Midi

1 box WG1201BOX Protein Gels, 1.0 mm, 12-well Protein Gels, 1.0 mm, 10-well Gels, 1.0 mm, 15-well
Protein Gels, 12+2-well NuPAGE Novex 38% Tris-Acetate 1 box EA03755BOX Novex 1020% Tris-Glycine Mini 1 box EC61352BOX Novex 18% Tris-Glycine Mini Protein 1 box EC6508BOX
NuPAGE Novex 10% Bis-Tris Midi

1 box WG1201A Protein Gels, 1.0 mm, 15-well Protein Gels, 1.0 mm, 12-well Gels, 1.5 mm, 10-well
Protein Gels, 12+2-well, w/adapters NuPAGE Novex 38% Tris-Acetate 1 box EA0378BOX Novex 1020% Tris-Glycine Mini 1 box EC61355BOX Novex 18% Tris-Glycine Mini Protein 1 box EC65085BOX
NuPAGE Novex 10% Bis-Tris Midi 1 box WG1202BOX Protein Gels, 1.5 mm, 10-well Protein Gels, 1.0 mm, 15-well Gels, 1.5 mm, 15-well
Protein Gels, 20-well NuPAGE Novex 38% Tris-Acetate 1 box EA03785BOX Novex 1020% Tris-Glycine Mini 1 box EC61385BOX Novex 4% Tris-Glycine Mini Protein 1 box EC6055BOX
NuPAGE Novex 10% Bis-Tris Midi

1 box WG1202A Protein Gels, 1.5 mm, 15-well Protein Gels, 1.5 mm, 15-well Gels, 1.0 mm, 10-well
Protein Gels, 20-well, w/adapters NuPAGE Novex 7% Tris-Acetate 1 box EA0355BOX Novex 12% Tris-Glycine Mini Protein 1 box EC6001BOX Novex 4% Tris-Glycine Mini Protein 1 box EC60552BOX
NuPAGE Novex 10% Bis-Tris Midi

1 box WG1203BOX Protein Gels, 1.0 mm, 10-well Gels, 1.0 mm, 1-well Gels, 1.0 mm, 12-well
Protein Gels, 26-well NuPAGE Novex 7% Tris-Acetate 1 box EA03552BOX Novex 12% Tris-Glycine Mini Protein 1 box EC6005BOX Novex 4% Tris-Glycine Mini Protein 1 box EC6058BOX
NuPAGE Novex 10% Bis-Tris Midi 1 box WG1203A Protein Gels, 1.0 mm, 12-well Gels, 1.0 mm, 10-well Gels, 1.5 mm, 10-well
Protein Gels, 26-well, w/adapters NuPAGE Novex 7% Tris-Acetate 1 box EA03555BOX Novex 12% Tris-Glycine Mini Protein 1 box EC60052BOX Novex 4% Tris-Glycine Mini Protein 1 box EC60585BOX
NuPAGE Novex 412% Bis-Tris Midi

1 box WG1401BOX Protein Gels, 1.0 mm, 15-well Gels, 1.0 mm, 12-well Gels, 1.5 mm, 15-well
Protein Gels, 12+2-well NuPAGE Novex 7% Tris-Acetate 1 box EA0358BOX Novex 12% Tris-Glycine Mini Protein 1 box EC60055BOX Novex 412% Tris-Glycine Mini Protein 1 box EC6035BOX
NuPAGE Novex 412% Bis-Tris Midi

1 box WG1401A Protein Gels, 1.5 mm, 10-well Gels, 1.0 mm, 15-well Gels, 1.0 mm, 10-well
Protein Gels, 12+2-well, w/adapters NuPAGE Novex 7% Tris-Acetate 1 box EA03585BOX Novex 12% Tris-Glycine Mini Protein 1 box EC6008BOX Novex 412% Tris-Glycine Mini Protein 1 box EC60352BOX
NuPAGE Novex 412% Bis-Tris Midi

1 box WG1402BOX Protein Gels, 1.5 mm, 15-well Gels, 1.5 mm, 10-well Gels, 1.0 mm, 12-well
Protein Gels, 20-well NuPAGE Tris-Acetate Midi Gels (8 cm x 8 cm) Novex 12% Tris-Glycine Mini Protein 1 box EC60085BOX Novex 412% Tris-Glycine Mini Protein 1 box EC60355BOX
NuPAGE Novex 412% Bis-Tris Midi

1 box WG1402A NuPAGE Novex 38% Tris-Acetate

1 box WG1601BOX Gels, 1.5 mm, 15-well Gels, 1.0 mm, 15-well
Protein Gels, 20-well, w/adapters Midi Protein Gels, 12+2W Novex 14% Tris-Glycine Mini Protein 1 box EC6485BOX Novex 412% Tris-Glycine Mini Protein 1 box EC6038BOX
NuPAGE Novex 412% Bis-Tris Midi

1 box WG1403BOX NuPAGE Novex 38% Tris-Acetate

1 box WG1601A Gels, 1.0 mm, 10-well Gels, 1.5 mm, 10-well
Protein Gels, 26-well Midi Protein Gels, 12+2W, w/adapters Novex 14% Tris-Glycine Mini Protein 1 box EC64852BOX Novex 412% Tris-Glycine Mini Protein 1 box EC60385BOX
NuPAGE Novex 412% Bis-Tris Midi

1 box WG1403A NuPAGE Novex 38% Tris-Acetate

1 box WG1602BOX Gels, 1.0 mm, 12-well Gels, 1.5 mm, 15-well
Protein Gels, 26-well, w/adapters Midi Protein Gels, 20W Novex 14% Tris-Glycine Mini Protein 1 box EC64855BOX Novex 420% Tris-Glycine Mini Protein 1 box EC6021BOX
NuPAGE Novex 8% Bis-Tris Midi

1 box WG1001BOX NuPAGE Novex 38% Tris-Acetate

1 box WG1602A Gels, 1.0 mm, 15-well Gels, 1.0 mm, 1-well
Protein Gels, 12+2-well Midi Protein Gels, 20W, w/adapters Novex 14% Tris-Glycine Mini Protein 1 box EC6488BOX Novex 420% Tris-Glycine Mini Protein 1 box EC6025BOX
NuPAGE Novex 8% Bis-Tris Midi

1 box WG1001A NuPAGE Novex 38% Tris-Acetate

1 box WG1603BOX Gels, 1.5 mm, 10-well Gels, 1.0 mm, 10-well
Protein Gels, 12+2-well, w/adapters Midi Protein Gels, 26W Novex 14% Tris-Glycine Mini Protein 1 box EC64885BOX Novex 420% Tris-Glycine Mini Protein 1 box EC60252BOX
NuPAGE Novex 8% Bis-Tris Midi

1 box WG1002BOX NuPAGE Novex 38% Tris-Acetate

1 box WG1603A Gels, 1.5 mm, 15-well Gels, 1.0 mm, 12-well
Protein Gels, 20-well Midi Protein Gels, 26W, w/adapters Novex 16% Tris-Glycine Mini Protein 1 box EC6495BOX Novex 420% Tris-Glycine Mini Protein 1 box EC60255BOX
NuPAGE Novex 8% Bis-Tris Midi 1 box WG1002A Novex Tris-Glycine Mini Gels (8 cm x 8 cm) Gels, 1.0 mm, 10-well Gels, 1.0 mm, 15-well
Protein Gels, 20-well, w/adapters Novex 10% Tris-Glycine Mini Protein 1 box EC6075BOX Novex 16% Tris-Glycine Mini Protein 1 box EC64952BOX Novex 420% Tris-Glycine Mini Protein 1 box EC60249BOX
NuPAGE Novex 8% Bis-Tris Midi

1 box WG1003BOX Gels, 1.0 mm, 10-well Gels, 1.0 mm, 12-well Gels, 1.0 mm, 9-well
Protein Gels, 26-well Novex 10% Tris-Glycine Mini Protein 3 boxes EC6075BX30 Novex 16% Tris-Glycine Mini Protein 1 box EC64955BOX Novex 420% Tris-Glycine Mini Protein 1 box EC6028BOX
NuPAGE Novex 8% Bis-Tris Midi

1 box WG1003A Gels, 1.0 mm, 10-well - Value Pack Gels, 1.0 mm, 15-well Gels, 1.5 mm, 10-well
Protein Gels, 26-well, w/adapters Novex 10% Tris-Glycine Mini Protein 1 box EC60752BOX Novex 16% Tris-Glycine Mini Protein 1 box EC6498BOX Novex 420% Tris-Glycine Mini Protein 1 box EC60285BOX
NuPAGE Tris-Acetate Mini Gels (8 cm x 8 cm) Gels, 1.0 mm, 12-well Gels, 1.5 mm, 10-well Gels, 1.5 mm, 15-well

NuPAGE Novex 38% Tris-Acetate 10 gels EA0375BOX Novex 10% Tris-Glycine Mini Protein 1 box EC60755BOX Novex 16% Tris-Glycine Mini Protein 1 box EC64985BOX Novex 6% Tris-Glycine Mini Protein 1 box EC6065BOX
Protein Gels, 1.0 mm, 10-well Gels, 1.0 mm, 15-well Gels, 1.5 mm, 15-well Gels, 1.0 mm, 10-well

Appendix
84 Appendix Protein gel electrophoresis technical handbook 85

Ordering information
Product Quantity Cat. No. Product Quantity Cat. No. Product Quantity Cat. No. Product Quantity Cat. No.

Novex 6% Tris-Glycine Mini Protein 1 box EC60652BOX Novex 420% Tris-Glycine Midi 1 box WT4201A Novex Tricine Gels Prepare samples and select buffers: SDS-PAGE
Gels, 1.0 mm, 12-well Protein Gels, 12+2-well, w/adapters Novex 10% Tricine Protein Gels,

1 box EC6675BOX Pierce SDS-PAGE Sample Prep Kit 50 89888
1.0 mm, 10-well reactions
Novex 6% Tris-Glycine Mini Protein

1 box EC60655BOX Novex 420% Tris-Glycine Midi

1 box WT4202BOX
Gels, 1.0 mm, 15-well Protein Gels, 20-well Novex 10% Tricine Protein Gels, 1 box EC66752BOX Bolt Transfer Buffer (20X) 125 mL BT0006
Novex Tris-Glycine Midi Gels (8 cm x 13 cm) Novex 420% Tris-Glycine Midi

1 box WT4202A 1.0 mm, 12-well Bolt Transfer Buffer (20X) 1L BT00061
Novex 10% Tris-Glycine Midi Protein 1 box WT0101BOX Protein Gels, 20-well, w/adapters Novex 16% Tricine Protein Gels, 1 box EC6695BOX 4X Bolt LDS Sample Buffer 10 mL B0007
Gels, 12+2-well Novex 420% Tris-Glycine Midi 1 box WT4203BOX 1.0 mm, 10-well
20X Bolt MES SDS Running Buffer 500 mL B0002
Novex 10% Tris-Glycine Midi Protein 1 box WT0101A Protein Gels, 26-well Novex 16% Tricine Protein Gels, 1 box EC66952BOX
Gels, 12+2-well, w/adapters 1.0 mm, 12-well 20X Bolt MES SDS Running Buffer 5L B0002-02
Novex 420% Tris-Glycine Midi 1 box WT4203A
Novex 10% Tris-Glycine Midi Protein 1 box WT0102BOX Protein Gels, 26-well, w/adapters Novex 16% Tricine Protein Gels, 1 box EC66955BOX 20X Bolt MOPS SDS Running Buffer 500 mL B0001
Gels, 20-well Novex 8% Tris-Glycine Midi Protein 1 box WT0081BOX 1.0 mm, 15-well 20X Bolt MOPS SDS Running Buffer 5L B0001-02
Novex 10% Tris-Glycine Midi Protein

1 box WT0102A Gels, 12+2-well Novex 1020% Tricine Protein Gels,

1 box EC6625BOX Bolt Antioxidant 15 mL BT0005
Gels, 20-well, w/adapters Novex 8% Tris-Glycine Midi Protein 1 box WT0081A 1.0 mm, 10-well
NuPAGE Tris-Acetate SDS Running 500 mL LA0041
Novex 10% Tris-Glycine Midi Protein 1 box WT0103BOX Gels, 12+2-well, w/adapters Novex 1020% Tricine Protein Gels, 1 box EC66252BOX Buffer (20X)
Gels, 26-well Novex 8% Tris-Glycine Midi Protein 1 box WT0082BOX 1.0 mm, 12-well
NuPAGE MOPS SDS Running Buffer 500 mL NP0001
Novex 10% Tris-Glycine Midi Protein 1 box WT0103A Gels, 20-well Novex 1020% Tricine Protein Gels, 1 box EC66255BOX (20X)
Gels, 26-well, w/adapters Novex 8% Tris-Glycine Midi Protein 1 box WT0082A 1.0 mm, 15-well
NuPAGE MOPS SDS Running Buffer 5L NP000102
Novex 12% Tris-Glycine Midi Protein

1 box WT0121BOX Gels, 20-well, w/adapters Novex IEF Gels (20X)
Gels, 12+2-well Novex 8% Tris-Glycine Midi Protein 1 box WT0083BOX Novex pH 37 IEF Protein Gels, 5 gelsbox EC66452BOX NuPAGE MES SDS Running Buffer 5L NP000202
Novex 12% Tris-Glycine Midi Protein

1 box WT0121A Gels, 26-well 1.0 mm, 12-well (20X)
Gels, 12+2-well, w/adapters Novex 8% Tris-Glycine Midi Protein 1 box WT0083A Novex pH 37 IEF Protein Gels, 5 gelsbox EC6645BOX NuPAGE MES SDS Running Buffer 500 mL NP0002
Novex 12% Tris-Glycine Midi Protein

1 box WT0122BOX Gels, 26-well, w/adapters 1.0 mm, 10-well (20X)
Gels, 20-well Novex 816% Tris-Glycine Midi Protein 1 box WT8161BOX Novex pH 310 IEF Protein Gels, 5 gelsbox EC6655BOX Novex Tris-Glycine SDS Running Buffer 4x1L LC26754
Novex 12% Tris-Glycine Midi Protein

1 box WT0122A Gels, 12+2-well 1.0 mm, 10-well (10X)
Gels, 20-well, w/adapters Novex 816% Tris-Glycine Midi Protein 1 box WT8161A Novex Zymogram Gels
Novex Tris-Glycine SDS Running Buffer 500 mL LC2675
Novex 12% Tris-Glycine Midi Protein

1 box WT0123BOX Gels, 12+2-well, w/adapters Novex 12% Zymogram (Casein) 1 box EC64052BOX (10X)
Gels, 26-well Novex 816% Tris-Glycine Midi Protein 1 box WT8162BOX Protein Gels, 1.0 mm, 12-well
Novex Tris-Glycine SDS Running Buffer 5L LC26755
Novex 12% Tris-Glycine Midi Protein

1 box WT0123A Gels, 20-well Novex 416% Zymogram (Blue Casein) 1 box EC6415BOX (10X)
Gels, 26-well, w/adapters Novex 816% Tris-Glycine Midi Protein 1 box WT8162A Protein Gels, 1.0 mm, 10-well
Novex Tricine SDS Running Buffer 500 mL LC1675
Novex 412% Tris-Glycine Midi Protein

1 box WT4121BOX Gels, 20-well, w/adapters Novex 12% Zymogram (Casein) 1 box EC6405BOX (10X)
Gels, 12+2-well Novex 816% Tris-Glycine Midi Protein 1 box WT8163BOX Protein Gels, 1.0 mm, 10-well
NuPAGE LDS Sample Buffer (4X) 10 mL NP0007
Novex 412% Tris-Glycine Midi Protein

1 box WT4121A Gels, 26-well Novex 10% Zymogram (Gelatin) 1 box EC61755BOX
Novex Tricine SDS Sample Buffer (2X)

20 mL LC1676
Gels, 12+2-well, w/adapters Novex 816% Tris-Glycine Midi Protein 1 box WT8163A Protein Gels, 1.0 mm, 15-well
Gels, 26-well, w/adapters Novex Tris-Glycine SDS Sample Buffer 20 mL LC2676
Novex 412% Tris-Glycine Midi Protein 1 box WT4122BOX Novex 10% Zymogram (Gelatin) 1 box EC61752BOX
(2X)
Gels, 20-well NativePAGE Gels Protein Gels, 1.0 mm, 12-well
Novex Tris-Glycine Transfer Buffer 500 mL LC3675
Novex 412% Tris-Glycine Midi Protein 1 box WT4122A NativePAGE Novex 312% Bis-Tris 1 box BN1001BOX Novex 10% Zymogram (Gelatin) 1 box EC6175BOX
(25X)
Gels, 20-well, w/adapters Protein Gels, 1.0 mm, 10-well Protein Gels, 1.0 mm, 10-well
E-PAGE High Throughput Gel System NuPAGE Transfer Buffer (20X) 125 mL NP0006
Novex 412% Tris-Glycine Midi Protein 1 box WT4123BOX NativePAGE Novex 312% Bis-Tris 1 box BN1003BOX
Gels, 26-well Protein Gels, 1.0 mm, 15-well E-PAGE 8% Protein Gels, 48-well 8 gels EP04808 NuPAGE Transfer Buffer (20X)

1L NP00061
Novex 412% Tris-Glycine Midi Protein

1 box WT4123A NativePAGE Novex 416% Bis-Tris

1 box BN1002BOX E-Holder Platform 2 units EH03 NuPAGE Antioxidant

15 mL NP0005
Gels, 26-well, w/adapters Protein Gels, 1.0 mm, 10-well
E-PAGE Loading Buffer 1 4.5 mL EPBUF01 Novex Tris-Glycine SDS Buffer Kit

1 kit LC2677
Novex 420% Tris-Glycine Midi 1 box WT4201BOX NativePAGE Novex 416% Bis-Tris 1 box BN1004BOX
E-PAGE 6% Protein Gels, 96-well 8 gels EP09606 NuPAGE MOPS SDS Buffer Kit

1 kit NP0050
Protein Gels, 12+2-well Protein Gels, 1.0 mm, 15-well
(for Bis-Tris Gels)
Daughter E-Base Device 1 unit EBD03
NuPAGE MES SDS Buffer Kit 1 kit NP0060
Mother E-Base Device

1 unit EBM03 (for Bis-Tris Gels)

Appendix
86 Appendix Protein gel electrophoresis technical handbook 87

Ordering information
Product Quantity Cat. No. Product Quantity Cat. No. Product Quantity Cat. No. Product Quantity Cat. No.

NuPAGE Tris-Acetate SDS Buffer Kit 1 kit LA0050 Select Protein Standards: Unstained Electrophoresis chamber systems and power supplies Coomassie stains
(for Tris-Acetate gels), Contains 1 ea. PageRuler Unstained Low Range 2 x 250 L 26632 Mini Gel Tank 1 unit A25977 PageBlue Protein Staining Solution 1L 24620
LA0041, NP0004, NP0005, NP0007 Protein Ladder
XCell SureLock Mini-Cell 1 unit EI0001 SimplyBlue SafeStain 1L LC6060
Novex Tricine SDS Buffer Kit, 1 kit LC1677 PageRuler Unstained Protein Ladder 2 x 250 L 26614
XCell4 SureLock Midi-Cell 1 each WR0100 SimplyBlue SafeStain 3.5 L LC6065
includes LC1676 & LC1675
NativeMark Unstained Protein Standard 5 x 50 L LC0725
PowerEase 90W Power Supply 1 each PS0090 Imperial Protein Stain

1L 24615
Pierce LDS Sample Buffer, Non- 5 mL 84788
Prestained (115 VAC)
Reducing (4X) Imperial Protein Stain

3x1L 24617
PageRuler Prestained Protein Ladder 2 x 250 L 26616 PowerEase 90W Power Supply 1 each PS0091
Pierce Lane Marker Non-Reducing 5 mL 39001 Silver stains
Sample Buffer PageRuler Prestained Protein Ladder 10 x 250 L 26617 (230 VAC)
Pierce Silver Stain Kit 1L 24612
Pierce 10X Tris-Glycine SDS Buffer 1L 28362 PageRuler Plus Prestained 2 x 250 L 26619 PowerEase 300W Power Supply 1 each PS0300
SilverXpress Silver Staining Kit 1 kit* LC6100
Protein Ladder (115 VAC)
BupH Tris-Glycine-SDS Buffer Packs

40 packs 28378 Pierce Silver Stain for 1L 24600
PageRuler Plus Prestained 10 x 250 L 26620 PowerEase 300W Power Supply 1 each PS0301
Native Electrophoresis Mass Spectrometry
Protein Ladder (230 VAC)
Novex Tris-Glycine Native Running 500 mL LC2672 *1 kit contains sufficient reagents to stain 25 mini gels
Spectra Multicolor Broad Range 2 x 250 L 26634
Buffer (10X)
Protein Ladder Fluorescent and specialty stains
Novex Tris-Glycine Native Sample 20 mL LC2673 SYPRO Orange Protein Gel Stain 500 L S-6650
Spectra Multicolor Broad Range 10 x 250 L 26623
Buffer (2X)
Protein Ladder SYPRO Orange Protein Gel Stain 10 x 50 L S-6651
NativePAGE Running Buffer (20X) 1L BN2001
HiMark Prestained Protein Standard

250 L LC5699 SYPRO Red Protein Gel Stain 500 L S-6653
NativePAGE Running Buffer Kit 1 kit BN2007
Spectra Multicolor High Range

2 x 250 L 26625 SYPRO Red Protein Gel Stain 10 x 50 L S-6654
NativePAGE Cathode Buffer Additive 250 mL BN2002 Protein Ladder
(20X) SYPRO Ruby Protein Gel Stain

1L S-12000
Western
NativePAGE Sample Buffer (4X) 10 mL BN2003 SYPRO Ruby Protein Gel Stain

200 mL S-12001
MagicMark XP Western 250 L LC5602
NativePAGE 5% G-250 Sample 0.5 mL BN2004 Protein Standard SYPRO Ruby Protein Gel Stain

5L S-21900
Additive Pro-Q Emerald 488 Glycoprotein

1 kit P-21875
MagicMark XP Western 50 L LC5603
NativePAGE Sample Prep Kit

1 kit BN2008 Protein Standard Gel Stain Kit

DDM (n-dodecyl -D-maltoside) (10%) 1 mL BN2005 Specialty Pro-Q Diamond Phosophoprotein 1L P-33300
Gel Stain Kit
Digitonin (5%) 1 mL BN2006 PageRuler Prestained NIR

2 x 250 L 26635
Protein Ladder Pro-Q Diamond Phosophoprotein 200 mL P-33301
Zymography Gel Stain Kit
BenchMark Fluorescent 125 L LC5928
Novex Zymogram Developing Buffer 500 mL LC2671
Protein Standard Pro-Q Diamond Phosophoprotein 5L P-33302
(10X)
Gel Stain Kit
BenchMark His-tagged

125 L LC5606
Novex Zymogram Renaturing Buffer 500 mL LC2670 Pierce Power Stainer
Protein Standard
(10X)
IEF Marker 310 500 L 39212-01 Pierce Power Stainer 1 unit 22833
IEF
Pierce Power Stainer Welcome Pack

1 unit 22833SPCL*
Novex IEF Anode Buffer (50X) 100 mL LC5300
*Welcome pack includes Pierce Power Station, Pierce Power Stain Cassette, Western Blot
Novex IEF Cathode Buffer pH 3-10 (10X) 125 mL LC5310 Roller, Power Cord with C/13 Connector, Quick Start Guide, Pierce Mini Gel Power
Staining Kit
Novex IEF Cathode Buffer pH 3-7 (10X) 125 mL LC5370
Novex pH 3-10 IEF Buffer Kit,

1 kit LC5317
Includes LC5300, LC5310, LC5311
Novex pH 3-7 IEF Buffer Kit, 1 kit LC5377
Includes LC5300, LC5370, LC5371
Novex IEF Sample Buffer pH 3-10 (2X) 25 mL LC5311
Novex IEF Sample Buffer pH 3-7 (2X)

25 mL LC5371

Appendix
Appendix
Crossword puzzle challenge
To participate in the crossword puzzle challenge,
Separate Crossword
go to thermofisher.com/pagecrossword
Complete the crossword below
1 2

6 7

10

11

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15

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Across Xcell4SureLock DownHermannSchagger UlrichLaemmli
ArneTiselius SheldonEngelhorn
5. Can you name one of the scientists who developed 1. Which tank is compatible with >180 mini gels? pg. 52
BisTris MagicMark
blue native polyacrylamide gel electrophoresis? pg. 17Unstained PowerStainer
2. Which Bolt minimizes
gel chemistry Minigeltank HarrySvennsonRilbe
protein degradation? pg. 9
6. Which tank is compatible with midigels? pg. 56 3. Who first published SDS-PAGE as a method for the
Prestained PowerEase NuPAGE
9. Which ladder can be used for accurate molecular analysis of cleavage of structural proteins in
weight estimation directly on western blots? pg. 46 bacteriophage? pg. 7
10. Can you name one of the scientists who filed a 4. Can you name one of the scientists who first
patent for the netural-pH Bis-Tris system in 1996? pg. 11 described the theory of separation of amphoteric
12. Which gel has a unique wedge well design that proteins along a pH gradient in the 1960s? pg. 21
allows you to load 2x the sample volume? pg. 10 7. What power supply is available for use with the Mini
13. Which protein ladder would you use for Gel Tank? pg. 58
approximate determination of molecular weight? pg. 35 8. What is a fast alternative to traditional Coomassie
15. Who won the Nobel Prize for analysis of serum staining? pg. 72
proteins by electrophoresis in 1948? pg. 5 11. Which ladder would you use for precise
determination of molecular weight? pg. 35
14. Which type of gel has been referenced in >20,000
publications? pg. 12

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