Practical Protein Chemistry - A. Darbre 1989

Affinity Chromatography of Proteins
Adsorption and Elution

Before using the affinity sorbent, it is recommended to wash it free of 0.02% sodium azide, which is typically added to commercial preparations to prevent biodegradation of the matrix. Protein or free Ligand impurities are removed by a brief wash with 1 M acetic acid, followed by thorough washing with a standard neutral buffer solution. Prior to sample loading, the specific activity of the target protein must be determined. It is advisable to measure protein concentration using an immunochemical method, such as enzyme-linked immunosorbent assay (ELISA) or radioimmunoassay (RIA).

As a rule, the Isolation and Purification of Membrane Proteins are carried out in the presence of detergents. The presence of low concentrations of digitonin or sodium dodecyl sulfate does not affect the efficiency of Affinity Chromatography. However, in each specific case, it is necessary to account for The Effect of the detergent on sorption, elution, and particularly on the biological activity assay used prior to sample loading. The Use of detergent-free buffers can lead to non-specific protein sorption on the sorbent due to aggregation, which significantly impacts the degree of purification.

After Sample application, the Column is washed first with a neutral buffer solution and then with 3 M NaCl. Biological activity is determined in the collected fractions. Dilution of the wash fractions should be avoided whenever possible, as the target substance may be detected in them. This may be caused by column overloading or improperly chosen loading conditions. In such cases, the obtained fractions are reloaded onto the column under the same or different conditions.

A critical factor in optimizing elution conditions is the Selection of the eluent composition for desorbing the bound protein. For instance, the free ligand can be used as an eluent. In practice, however, this desorption option is rarely used due to the low solubility of ligands in aqueous buffers or their high cost. Moreover, in cases of non-specific binding, elution with a free ligand can yield erroneous results. More efficient desorption is achieved using ligands of different structures but with analogous Specificity. Enzyme Inhibitors or Cofactors, haptens corresponding to fragments of the antigen immobilized on the sorbent, hormone receptor antagonists, etc., are commonly employed as eluents.

In some cases, linear or step gradients of increasing eluent concentration are used to separate protein components exhibiting different affinities for the affinity sorbent. To desorb proteins with strong affinity for the sorbent driven by hydrophobic, electrostatic, or other interactions, harsher elution conditions are recommended, such as 1 M acetic acid, 6 M urea, 4 M guanidine-HCl, or 2 M iron(III) thiocyanate. However, such conditions cause the desorption of non-specifically bound proteins as well as the Denaturation of the target protein and the loss of its biological activity. Under these circumstances, immunochemical identification Methods should be employed. Another drawback of harsh elution conditions relates to a reduction in the total number of chromatographic cycles that can be performed on the given affinity sorbent. If proteins are eluted under strongly denaturing conditions, the sample must be immediately separated from the denaturing mixture by dialysis. When acid desorption is used, a minimal amount of acid should be applied, and the fractions must either be neutralized immediately or collected into tubes containing a pre-calculated volume of concentrated neutral phosphate buffer.

To evaluate the efficiency of affinity chromatography, the recovery of the target protein at each elution stage is of paramount importance. Therefore, all collected fractions should be retained until the experiment is completed. Protein recovery can vary over a wide range. The addition of a carrier protein increases the yield of biologically active material, but may also affect the degree of purification. Leaching of the ligand from the sorbent results in a decreased recovery of the bound protein or an apparent decrease in the yield of the isolated biologically active material. The extent of ligand Cleavage is assessed by measuring the ligand content in the wash fractions obtained prior to sample application [8], or by tracking the release of a radioactive label when radiolabeled ligands are used.

The presence of protease contaminants in the sample may hinder the Determination of the biological activity of the target proteins. The Interference of the ligand can be eliminated either by sample dilution or by exhaustive dialysis. However, this stage may carry the risk of Protein Denaturation. To prevent protein degradation, all operations—including sample application, sorption, washing, and elution—should be performed in the presence of protease inhibitors.