Affinity Chromatography with Immobilized Dyes

Abstract

Affinity chromatography is a very attractive concept for protein purification in that it exploits the one property that distinguishes each protein, namely, its ability to complex one or a small number of molecules (bioligands) with high affinity. Unfortunately, this attractive concept has several practical limitations that restrict its usefulness. Firstly, a bioligand must be covalently attached (immobilized) to the Chromatographic matrix in a manner which does not seriously diminish the affinity of the biofunctional site of the protein for the bioligand. Such attachment often requires execution of some adroit chemistry requiring either technical skills beyond that of a typical investigator or else adequate financial resources to purchase such a product. Secondly, a given immobilized bioligand will contribute to the purification of only a single or at best a small number of related proteins. Thus, a different affinity column must be available for each of the purified proteins required by an investigator. Thirdly, crude protein mixtures likely contain enzymes which can catalyze the hydrolysis of immobilized ligands, rendering them ineffective. Accordingly, affinity chromatography is commonly performed late in a purification scheme, where the concentrations of such hydrolytic enzymes are minimized by prior purification steps. Relegation of affinity chromatography to a late step in purification diminishes its selective potential.