Lab No.

4: Affinity Chromatography
(Week of October 27th /2008)

Task
i. Laboratory Introduction
A. Equilibration of Ni-Agarose matrix
B. Affinity Chromatography-Batch Method
C. Washing and Elution of PTEN-His Protein
D. Quantitation of Purification Yield by Bradford Assay
E. Purity Analysis by SDS/PAGE

By the successful completion of this lab you should be able to:

a) Explain the scientific principles and methodology employed in the isolation of a
protein which contains a poly histidine tag using affinity chromatography,
b) Explain how your protein interacts with the matrix at all steps within this
procedure,
c) Explain how your protein of interest is eluted off of the Ni-agarose resin

(a) Background:
Principles of Affinity Chromatography:

In this lab, you will be employing the technique of affinity chromatography to purify PTEN-
His from bacterial lysates. The principle of affinity chromatography is based on the
exploitation of the specific, reversible binding between the protein of interest and a specific
molecule immobilized on an inert support. This technique offers high selectivity, hence high
resolution, and high binding capacity for the protein of interest. Affinity chromatography is
unique in purification technology since it is the only technique that enables the purification
of a biomolecule on the basis of its biological function or individual chemical structure.
Purification that would otherwise be time-consuming, and complex using other techniques
can often be easily achieved utilizing affinity chromatography in a single step. The technique
can be used to isolate substances present at low concentration in large volumes of crude
sample, while not disturbing the native three dimensional fold of the protein.
 Protein Attraction:
The biological interactions between ligand and target molecule can be a result of electrostatic
or hydrophobic interactions, van der Waals' forces and/or hydrogen bonding. Therefore, to
elute the target molecule from the affinity matrix these interactions are reversed, either
specifically using a competitive ligand, or non-specifically, by changing the pH, ionic strength
or polarity.

Applications:
Successful affinity purification requires a biospecific ligand that can be covalently attached to
a chromatography matrix. The coupled ligand must retain its specific binding affinity for the
target molecules and, after washing away unbound material, the binding between the ligand
and target molecule must be reversible to allow the target molecules to be removed in an
active form. Any component can be used as a ligand to purify its respective binding partner.
Some typical biological interactions, frequently used in affinity chromatography, are listed
below.

Ligand Target
Enzyme Substrate analogue, inhibitor, cofactor
Antibody Antigen, virus, cell (epitopes)
Lectin Polysaccharide, glycoprotein ,cell surface receptor
Nucleic acid complementary base sequence, histones, nucleic acid polymerase,
nucleic acid binding protein
Hormone or vitamin Receptor or carrier protein
Metal ions Poly (His) fusion proteins
Glutathione Glutathione-S-transferase or GST fusion proteins

(1) Affinity matrix is equilibrated in binding buffer; (2) sample is applied under experimental conditions that favour
specific binding of the target molecule; (3) target protein is recovered by changing the condition to favour elution
of the bound molecule; (4) affinity matrix is re-equilibrated in binding buffer. (Photo courtesy of Amersham
Biosciences ©)

The matrix
The matrix is an inert support to which a ligand can be directly or indirectly coupled. The list
below highlights many of the properties required for an efficient and effective
chromatography matrix.
 • Extremely low non-specific adsorption, essential since the success of affinity
chromatography relies on specific interactions.
• Hydroxyl groups on the sugar residues are easily derivatized for covalent attachment
of a ligand, providing an ideal platform for the development of affinity media.
• An open pore structure ensures high capacity binding even for large biomolecules,
since the interior of the matrix is available for ligand attachment.
• Good flow properties for rapid separation.
• Stability under a range of experimental conditions such as high and low pH,
detergents and dissociating agents.

Spacer arms
The binding site of a target protein is often located deep within the molecule and an affinity
medium prepared by coupling small ligands, such as enzyme cofactors, directly to the matrix
may exhibit low binding capacity due to steric interference i.e. the ligand is unable to access
the binding site of the target molecule. In these circumstances a "spacer arm" is interposed
between the matrix and the ligand to facilitate effective binding. Spacer arms must be
designed to maximize binding, but to avoid non-specific binding effects. The improvement
that can be seen in purification as the spacer arm creates a more effective environment for
binding.

Elution:
There are typically four basic methods for the elution of your protein, all which involve the
weakening of the interaction between the functional group on the matrix and your protein:
(1) change buffer composition; (2) change pH; (3) competition for binding with target; or (4)
add substance which competes for binding to the immobilized ligand (functional group) –
this is the most common method of affinity chromatography and what will be used in
this lab.

Ni-NTA Affinity Chromatography:

The purification of recombinant proteins can often be simplified by incorporating a peptide
or protein tag of known size into a protein of interest. These “tags” are used due to their
high affinity for an immobilized column support. As well as providing a marker for
expression and facilitating detection of the recombinant protein, an important role for the
tag is to enable rapid purification by affinity chromatography. The two most commonly used
tags are glutathione-S-transferase (GST) and 6 x histidine residues (His)6. For this lab, the
fusion protein contains 6 x histidine residues (His)6 is located on the C-terminus of PTEN.

The (His) 6 tag is one of the most common tags used to facilitate the purification and
detection of recombinant proteins with a simple, one step purification procedure. The tag at
pH 8.0 is uncharged and relatively small in size and therefore it does not generally affect
secretion, compartmentalization, or folding of the fusion protein within the cell.

Proteins and peptides containing amino acids that have an affinity for metal ions can be
separated using immobilized metal affinity chromatography (IMAC). The metals are
immobilized onto a chromatographic medium by chelation. Certain amino acids, e.g.
histidine and cysteine, form complexes with the chelated metals around neutral pH (pH 6–
8). IMAC is excellent for purifying recombinant (His) 6 fusion proteins as well as many
natural proteins. This matrix is made by coupling a metal chelate forming ligand
(iminodiacetic acid) to agarose.

Before use, the matrix is loaded with a solution of divalent metal ions such as Ni2+, Zn2+,
Cu2+, Ca2+, Co2+ or Fe2+. The binding reaction with the target protein is pH dependent and
bound sample is, most commonly, eluted by reducing the pH and increasing the ionic
strength of the buffer or by including EDTA or imidazole in the buffer. In this lab, we will
be using a nickel-nitrilotriacetic acid (Ni-NTA) metal-affinity chromatography
matrix for the isolation of PTEN-His and elution will be performed using imidazole
buffer.

A B C D E

Interactions between polyhistidine tag (A) and Qiagen's Ni-NTA resin (B-E); A = poly-histidine tag; B = metal
(Ni2+); C & D = linker; and E = agarose bead.

A. Equilibration of Ni-Agarose Matrix

A1. From the front of the lab, acquire a 15 ml tube containing 200 µl of Ni-agarose
slurry. In the benchtop centrifuge, spin down the matrix at 5000 rpm for 2 minutes.

A2. Remove the supernatant and transfer it to waste. To the resin add 3 ml of Wash
Buffer A and mix by inversion.
A3. Again, in the benchtop centrifuge, spin down the matrix at 5000 rpm for 2 minutes.
Remove the supernatant and transfer to waste.

A4. Repeat steps A2-A3 once.

End of protocol A

Protocol B. Affinity Chromatography-Batch method

B1. From the front of the lab, acquire your labeled 50 ml tube containing the soluble
lysate of His-PTEN isolated in Week 2 and place on ice.

B2. At your workstation, add 10 ml of your PTEN-His lysate to the 15 ml tube

containing your equilibrated Ni-agarose matrix.

B3. Take your tube containing lysate and matrix to the rocking station located in the cold
room and incubate the chromatography matrix and lysate for 1.5 hrs at 4 °C. While
your sample is incubating, make one SDS/PAGE gel for purification analysis.

End of protocol B:

C. Washing and Elution of PTEN-His

C1. Spin down your sample and matrix in a swinging bucket rotor at 4000 rpm for 2 min.
Transfer 1 ml of the supernatant into a fresh microfuge tube labelled “Flow
through” fraction and place on ice. The remainder of the supernatant can be
discarded to waste.

C2. To your matrix add 3 ml of Wash Buffer A and invert five times to wash the resin.
Spin again as in step C1. Transfer 1 ml of the supernatant into a fresh microfuge
tube labelled “Wash 1” and place on ice. The remainder of the supernatant can be
discarded to waste.

C3. Repeat step C2 one more time.

C4. Repeat step C2 two more times except use Wash Buffer B (contains no detergent).

C5. Elute PTEN-His by incubating the matrix with 200 µl of Elution Buffer on ice for
15 minutes. Occassionally flick the bottom of the tube to facilitate elution. Spin
down the beads as in step C1. Transfer the supernatant into a fresh microfuge tube
labelled “Elution 1”.
C6. Repeat step C5 one more time labelling the tube “Elution 2”.

End of protocol C:

D. Protein Quantification by performing a Bradford Assay

D1. Determine and record the protein concentration of the various samples to be loaded
on the SDS/PAGE gel. The samples include the following: The Flow through
fraction, Wash 1, Wash 4, Elution 1, and Elution 2.

E. Analysis of Protein Purity by SDS/PAGE

E1. Analyze the purification procedure by mixing 16 ul of each sample mentioned in

section D with 4 ul of the SDS/PAGE loading dye and loading the samples into the
prepared SDS/PAGE gel.
E2. Stain the SDS/PAGE gel with Coomassie Blue stain and destain with the
ethanol:acetic acid buffer.

E3. Document your SDS/PAGE gel using the BioImager system.