Practical Protein Chemistry - A. Darbre 1989

Affinity Chromatography of Proteins
Applications of Affinity Chromatography

At what stage of the general purification scheme should Affinity Chromatography be used? The various potential Applications of affinity chromatography are shown schematically in Fig. 5.2.

Affinity chromatography can be used at the initial stage of purification, provided that the target protein interacts with the Ligand so strongly and specifically that Separation from accompanying impurities is achieved in a single step. Examples include the isolation of specific Antibodies from Blood serum using immobilized Antigens, or the extraction of Hormones from solubilized Plasma Membranes. Affinity chromatography is particularly effective as an initial step when the Stability of the target substance is questionable and the number of purification steps must be minimized. The cause of instability may be the thermolability of the protein molecule, sensitivity to proteases present in the crude extract, or the necessity of preserving a specific microenvironment—such as Phospholipids or other substances associated with the protein.

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FIG. 5.1. MAIN STAGES OF affinity chromatography.

Affinity chromatography is frequently employed with great success at The final stage of isolation, following partial purification by less specific, routine Methods such as ammonium sulfate precipitation, Gel filtration, and Ion-exchange chromatography. In this case, the specific elution of the target product can serve as a criterion of its purity, as well as a test for the retention of biological activity.

Affinity chromatography may also be used as an intermediate isolation step, although in this case achieving complete purification is not the primary objective.