11 - Affinity Chromatography

Abstract

This chapter provides an overview of affinity chromatography. It is a method of fractionation which exploits the biospecific binding of a particular molecule to a second molecule, often termed the “ligand”. The technique is extremely powerful. Purification factors of 2,000-20,000-fold are often possible, and it is sometimes possible to achieve purification to homogeneity in a single step. Affinity chromatography is a very common method for purification of low-concentration proteins, and the eluted protein is well suited for amino acid sequencing. The advent of monoclonal antibodies has revolutionized affinity chromatography. The presence of a charged linkage tends to cause nonspecific ionexchange effects which could degrade the specificity of the gel when used for affinity chromatography. This ion-exchange effect is most pronounced at very low salt concentrations, and it is overcome by performing the chromatography in buffers of high salt concentration. Affinity chromatography on antibody columns is divided into five equally important phases: sample preparation, precycling, binding, washing, and elution.