Affinity Chromatography

Affinity chromatography is a convective analytical or preparative technique which is used to separate components in a mixture of chemical compounds based on differences in their ability to bind to particular substrate. In affinity chromatography, a substrate is immobilized on the stationary phase media which is packed into a chromatography column. Mixtures containing the analyte are injected onto the column, where any components of the mixture with a propensity to bind to the substrate will become attached to the stationary phase and all other components of the mixture will simply run through the column. Attached particles can then be eluted from the column by adding a compound which disrupts the interaction between the substrate and the stationary phase. Commonly-used substrate ligands include metal-ions, antibodies, or small molecules such as ATP.

With the recent focus on recombinant proteins and antibodies in biomedical research, affinity chromatography has emerged as an important technique for the purification and analysis of therapeutics. Recombinant proteins are frequently engineered with affinity tags, such as a 6x His tag, or a GST tag. These tags will bind to nickel ions and glutathione respectively, both of which can be conjugated to a column matrix. This allows highly specific capture of the proteins from a cell lysate to aid in purification. Once captured, proteins can be eluted from nickel columns with imidazole, and GST columns with glutathione, allowing recovery of folded protein. Additionally, it has become common to conjugate the antibody of interest to a chromatography column matrix. This technique allows investigators to identify and/or purify antigens to any antibody. A variety of techniques can then be used to break the interaction between the antibody and antigen and elute the compound of interest from the column. However, these techniques often lead to unfolding and deactivation of the compound of interest, and the resulting damage to the analyte can require powerful analytical tools to detect and eliminate.

Here at Analytical Ventura we specialize in developing elution protocols which maximize the recovery of folded active protein. We offer services to assist you in purifications using commercially available affinity columns, or we can develop a custom affinity column for you. We have considerable experience in the use and creation of affinity chromatography strategies for purification of proteins and other molecules. Our scientists are happy to develop affinity chromatography protocols for your research, and/or novel purification strategies.

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