Practical Protein Chemistry - A. Darbre 1989

Affinity Chromatography of Proteins
Methods for Preparing Affinity Sorbents
Support Materials

The matrix must be resistant to biological and chemical agents. Commonly used matrices include agarose, Cellulose, polyacrylamide gels, and porous Glass (silica).

5.3.2.1. Agarose. Agarose is a linear polysaccharide composed of D-galactose and 3,6-anhydro-L-galactose residues. It is the most widely used matrix for Affinity Chromatography due to a favorable set of properties; in particular, agarose is quite stable under standard conditions. Furthermore, it is commercially available as highly standardized preparations.

Ligands and spacer arms are readily attached to agarose following Cyanogen bromide activation. A number of modifications to this Procedure are described in the literature. Cyanogen bromide activation is typically carried out in a buffer solution or in dimethylformamide. Throughout the activation process, the pH is maintained between 9 and 10 by continuous titration with alkali or a concentrated buffer. For laboratories unequipped to handle toxic cyanogen bromide (i.e., lacking dedicated fume hoods and vacuum systems), cyanogen bromide-activated agarose is commercially available. However, this commercial product is not without limitations. Several notable drawbacks include:

1) Biodegradation. Agarose is readily degraded by hydrolytic Enzymes produced by Bacteria present in the test solutions. A 0.02% sodium azide solution is commonly used as a protease inhibitor.

2) Chemical degradation. There is evidence that the covalent linkage between the matrix and the spacer arm or Ligand can be hydrolyzed by amines, such as in the presence of Tris-containing buffers. The affinity sorbent may also degrade under acidic conditions during the elution of tightly bound Proteins.

3) Nonspecific sorption. This drawback stems from the activation method. Cyanogen bromide Treatment activates numerous hydroxyl groups, not all of which are accessible to ligand molecules. Residual unreacted hydroxyl groups are typically blocked using an excess of a primary amine, Lysine, or ethylenediamine. Despite this, nonspecific binding may still occur. Non-covalent sorption is also observed when concentrated protein solutions—such as sera, Cell extracts, or seed extracts—are applied to the Column. To minimize nonspecific sorption, washing the sorbent with high-ionic-strength salt solutions is recommended, though this may inadvertently cause the desorption of proteins with low affinity for the ligand.

5.3.2.2. Cellulose. Due to its lower nonspecific sorption and the availability of straightforward ligand immobilization Methods, this heterogeneous glucose polymer is frequently employed for the preparation of immunosorbents. However, the hydrodynamic properties of cellulose are inferior to those of other matrices.

5.3.2.3. Polyacrylamide. The main advantage of polyacrylamide is its low nonspecific protein sorption. Nevertheless, difficulties may arise during the Introduction of spacer arms or subsequent ligand coupling.

5.3.2.4. Porous glass. Porous glass with various particle sizes and pore dimensions is commercially available. While it exhibits excellent mechanical strength and permeability, it suffers from insufficient chemical resistance, particularly in alkaline environments. In addition, nonspecific sorption is frequently observed during chromatography on porous glass Supports.

5.3.2.5. Insoluble proteins. One of the earliest types of affinity sorbents was a polymer prepared by adding ethyl chloroformate to a protein solution containing the ligand. Upon stirring the reaction mixture with a magnetic stirrer, an insoluble gel precipitates as fine particles [1]. To improve its hydrodynamic performance, the gel can be blended with a coarse fraction of Sephadex G-75.