Affinity chromatography: general methods

Affinity chromatography is one of the most diverse and powerful chromatographic methods for purification of a specific molecule or a group of molecules from complex mixtures. It is based on highly specific biological interactions between two molecules, such as interactions between enzyme and substrate, receptor and ligand, or antibody and antigen. These interactions, which are typically reversible, are used for purification by placing one of the interacting molecules, referred to as affinity ligand, onto a solid matrix to create a stationary phase while the target molecule is in the mobile phase. Successful affinity purification requires a certain degree of knowledge and understanding of the nature of interactions between the target molecule and the ligand to help determine the selection of an appropriate affinity ligand and purification procedure. With the growing popularity of affinity purification, many of the commonly used ligands coupled to affinity matrices are now commercially available and are ready to use. However, in some cases new affinity chromatographic material may need to be developed by coupling the ligand onto the matrix such that the ligand retains specific binding affinity for the molecule of interest. In this chapter, we discuss factors which are important to consider when selecting the ligand, proper attachment chemistry, and the matrix. In recent years, matrices with unique features which overcome some of the limitations of more traditional materials have been developed and these are also described. Affinity purification can provide significant time savings and several hundred-fold or higher purification, but the success depends on the method used. Thus, it is important to optimize the purification protocol to achieve efficient capture and maximum recovery of the target.