Affinity Chromatography

What is Affinity Chromatography???Affinity Chromatography is a method fromwhich we can separate biochemical mixtures.The separation is based on highly specific biological interaction.Examples:- Antigen and Antibody, Enzyme and Subtract• By the use of Affinity Chromatography we can separate proteins

on the basis of reversible interaction between protein and specific ligand.

• This technique offers high selectivity, high resolution and high capacity for the protein.• Affinity Chromatography is unique purification technology

Common terms in Affinity Chromatography

• Matrix: for ligand attachment. Matrix should be chemically and physically inert.

• Spacer arm: used to improve binding between ligand and target molecule by overcoming any effects of steric hindrance.

• Ligand: molecule that binds reversibly to a specific target molecule or group of target molecules.

• Binding: buffer conditions are optimized to ensure that the target molecules interact effectively with the ligand and are retained by the affinity medium as all other molecules wash through the column.

• Elution: buffer conditions are changed to reverse (weaken) the interaction between the target molecules and the ligand so that the target molecules can be eluted from the column

• Wash: buffers that wash unbound substances from the column without eluting the target molecules or that re-equilibrate the column back to the starting conditions

• Ligand coupling: covalent attachment of a ligand to a suitable pre-activated matrix to create an affinity medium

• Pre-activated matrices: matrices which have been chemically modified to facilitate the coupling of specific types of ligand.

Principle of Affinity Chromatography

• Inject a sample into an initially equilibrated affinity chromatography column

• Only the substances with affinity for the ligand will retained in the column.

• Other substances with no affinity for the ligand will eluted from the column.

• The substances retained in the column can be eluted from the column by changing pH or salt or organic solvent.

Method of Affinity Chromatography• Binding of the selected ligand to the matrix requires that a covalent

bond be formed between the two which is facilitated by derivitization of the sugar residues' hydroxyl groups.

• It is important to realize that the substrate might not be able to reach the ligand active site if it is hidden deep within the ligand. Therefore, most ligands are attached first to spacer arms which are then bonded to the matrix.

• The ligand-matrix gel is then loaded into an elution column.• Once the column has been prepared, the mixture containing isolate

is poured into the elution column. • Once in the column, gravity pulls the solution through the gel,

because most of the proteins do not bind to the ligand-matrix complex.

• However, when the ligand's recognized substrate passes through the gel, it binds to the ligand-matrix complex, halting its passage through the gel.

• Some of the impurities flow through the gel due to gravity, but most remain, unbound, in the gel column.

• In order to remove these unbound impurities, a wash of extreme pH, salt concentration, or temperature is run through the gel.

• It is important to use a strong wash so that all the impurities are removed, but it is also just as crucial that the wash be not so strong that it removes the bound isolates.

• Once the impurities are washed-out, the only remaining part of the protein mixture should be the desired isolates.

• Finally to collect your favorite isolate, which is still bound to the ligand-matrix in the gel, a stronger second wash is run through the column.

• This second wash relies on the reversible binding properties of the ligand, which allows the bound protein to dissociate from its ligand in the presence of this stronger wash.

• The protein is then free to run through the gel and be collected.

The Chromatogram• Affinity chromatography is not just limited to isolating one

protein; given a sample of similar proteins with similar binding affinities, a chromatogram can be generated.

• A chromatogram is a plot of absorbance vs. time for the elution of proteins in affinity chromatography.

• Four things may be learned from a chromatogram: 1) The level of complexity of the sample (indicated by the

number of peaks)2) Qualitative information about the sample composition (by

comparing peak positions with known standards) 3) Quantitative information of the relative component

concentrations (by comparing peak areas) 4) Total column performance (by comparing with known

standards).

Chromatogram

Applications of Affinity Chromatography

Affinity chromatography can be used to:• Purify and concentrate a substance from a

mixture into a buffering solution• Reduce the amount of a substance in a

mixture• Recognize what biological compounds bind to

a particular substance• Purify and concentrate an enzyme solution.

Case Studies.• Determination of binding constants

by affinity chromatography• In the use of affinity chromatography to characterize biospecific

interactions in terms of reaction stoichiometry and equilibrium constant. In that regard, the biospecificity incorporated into the design of the experiment ensures applicability of the method regardless of the sizes of the reacting solutes.

• By the adoption of different experimental strategies (column chromatography, simple partition equilibrium, solid-phase immunoassay and biosensor technology protocols) quantitative affinity chromatography can be used to characterize interactions governed by an extremely broad range of binding affinities.

• The link between ligand-binding studies and quantitative affinity chromatography is illustrated by means of partition equilibrium studies of glycolytic enzyme interactions with an exercise which emphasizes that the same theoretical expressions apply to naturally occurring examples of affinity chromatography in the cellular environment.

(Ref: Journal of Chromatography A Volume 1037, Issues 1-2, 28 May 2004, Pages 351-367)

• Experimental studies on affinity chromatography in an electric field

• A multi compartment electrolyzer, which has been used for preparative electrophoresis [Z. Liu, Z. Huang, J.-Y. Cong, et al., Sep. Sci. Technol. 31 (1996) 427], is applied for carrying out affinity chromatography in an alternating electric field.

• The effect of electric field strength on the adsorption and desorption characteristics is experimentally examined with human serum albumin and Blue Sepharose Fast Flow as a model system.