Affinity chromatography

Affinity chromatography is a chromatographic separation process for isolating an analyte from a solution of various substances. This requires that a suitable ligand ( binding partner ) to the analyte of interest (protein) is provided. It is one of the most powerful separation methods. However, the columns used are relatively expensive, so that the method is used only in special cases or small-scale ( lab-scale ).

Principle

The separation is usually done in columns, but can also be performed in batch mode. The purification effect of this method is based either on the specific recognition of a protein by an antibody, or for enzymes for use of the specific affinity of an enzyme to an inhibitor, substrate or cofactor.

The stationary phase, often a gel, for example, dextrans or cross-linked agarose ( Sepharose ® trade name ), is (for example, antibody) coupled with an appropriate ligand that specifically binds the analyte to be purified. It must be ensured in practice that the affinity for the analyte is not too high, because the elution is difficult. Conversely, for the preparative purification of antibodies ( immunoglobulins ) and a stationary phase with a protein (usually protein A, G or L ) are used, which binds certain immunoglobulin classes.

For binding of a ligand to the support material before it is converted into an activated form. In the example of agarose, the cyanogen bromide activation method is contemplated to be generated in the reactive imidocarbonate groups which react with amino groups of analytes to form a covalent bond. After fixation, a low molecular weight ligands on the matrix (eg agarose activated ) this may lead to steric hindrance when the analyte has a large molecular size. In this case, the ligand may be coupled to the matrix via a bridge member ( spacer). As spacers normally short hydrocarbon chains are used, then to some extent protrude from the matrix surface.

For the binding of the ligand and the target protein is the association constant, derived from the equilibrium constant, is important. The larger this is, the cheaper passes by affinity separation. The value should be at least Kass = 104 mol -1.

Implementation

To perform affinity chromatography work today usually biocompatible HPLC systems are used, but these are operated here only bar with lower pressures of up to 150. The mixture to be separated is fed into a rule about sample loop onto the column and the substance of interest is bound by the ligand. All other components exit the column quickly, since they do not interact strongly with the ligand. After a washing step to remove non-specifically bound impurities, the bound on the ligand analyte is brought by changing the conditions ( buffer composition ) to likewise leave the column ( elution). The eluent used an acidic buffer or a solvent / water mixture is often used. Alternatively competitively to the target protein active substances and an excess of free ligand can be added. The eluate containing the purified and enriched analytes.

Examples

Examples of ligands for protein purification: