Protein Purification by Affinity Chromatography

The preparation of a number of agarose and polyacrylamide bead derivatives useful in the purification of proteins by affinity chromatography is described. These techniques permit (a) the attachment of ligands to the gel through extended hydrocarbon chains which place the ligand at varying distances from the gel matrix backbone; (b) the covalent attachment of ligands to agarose or polyacrylamide gels through amino, carboxyl, phenolic, or imidazole groups of the ligand; and (c) the preparation of adsorbents containing ligands attached by bonds which are susceptible to specific chemical cleavage, thus providing means of removing the intact protein-ligand complex from the affinity adsorbent. It is demonstrated that successful application of affinity chromatography in many cases will critically depend on placing the ligand at a considerable distance from the matrix backbone. Techniques are also described which provide important approaches and considerations in the insolubilization of peptides and proteins to agarose and polyacrylamide.