Affinity Chromatography is based on the principle of biological recognition. Examples
of biological recognition include antibody-antigen, enzyme-substrate, and receptor-agonist
binding interactions. These interactions typically have high affinity
(Kd < 10-6 M), yet are reversible when conditions are changed. Due to
the specificity of this recognition, it is often possible to obtain 100- or even 1000-fold
increases in purity of the protein sample.
Steps in affinity chromatography
The first step in affinity chromatography is to prepare a stationary phase by immobilizing
one of the two recognized components on an insoluble hydrophobic polymer such as agarose.
A crude mixture containing the other recognized component is then applied to this
During a wash step, which often includes a high concentration of salt to overcome weaker
recognition interactions, all proteins except those that bind tightly to the stationary
phase are eluted.
The wash is followed by an elution step in which a soluble form of the immobilized ligand
is used to displace the bound protein from the stationary phase. It may also be possible
to release the bound protein by changing the pH of the buffers in the mobile phase.
There are thousands of examples of affinity chromatography in the literature, including
the purification of trypsin and other serine proteases using immobilized bovine pancreatic
trypsin inhibitor as the stationary phase.