Protein Elution in Affinity Chromatography- Welch Materials, Inc.

Protein Elution in Affinity Chromatography

Affinity chromatography is a kind of special chromatography technology which uses the specific affinity between the substance to be separated and the ligand to achieve separation according to the specific interaction between biological molecules. Such as antigen and antibody, DNA and RNA, enzyme and substrate or inhibitor, etc.

There are two types of elution methods, namely, specific elution and non-specific elution.

Specificity of elution process, can choose a substance and strong affinity ligand to join the eluent, the substance and stay separated competition of ligand, under appropriate conditions, such as the concentration of the substance with strong affinity ligand or larger, ligands are basic was dominated by the material, the original and the ligand binding to separation material be replaced out of ligand, So that it’s eluted off; Also can choose a kind of isolation material has strong affinity for material to join the eluent, this kind of material with ligand to treat separation material, the combination of competition in under appropriate conditions, such as the substance isolation material stay strong affinity or concentration is larger, stay separated material is basic and out of the ligand binding by this kind of material, which were cleared off. Specific elution is usually mild and is more likely to preserve protein activity. The disadvantages of this method are slow elution, peak width, and the need to remove the competing medium from the recovered protein.

For non-specific elution, elution conditions should be optimized according to the interaction mechanism between ligand and protein, such as increasing salt concentration, decreasing ionic interaction or changing protonation/ionization state by changing pH, so as to adjust hydrogen bond strength, hydrophobic interaction and electrostatic interaction. For example, an antibody can be eluted from a fixed Protein G by changing the pH, but the affinity of Protein G to the antibody is so strong that different combinations of elution conditions are required to achieve maximum antibody release.