Affinity chromatography immobilization methods

Although affinity chromatography has not been used directly as an analytical method, it may be modified in the future to produce a viable technique. Leucovorin has been used as an effective spacer in obtaining active samples of dihydrofolate reductase.79 If the enzyme could be immobilized without losing its activity, perhaps it could be used to separate folates. 

A third item to consider in using affinity chromatography is the way in which the ligand is attached to the solid support, or the immobilization method. Several techniques are available for this, including both covalent and noncovalent coupling methods [25,36]. For a protein or peptide, this generally... 

Table 13.3 summarizes various covalent immobilization methods that are used in affinity chromatography. Each of these methods involves at least two steps (1) an activation step, in which the support is converted to a form that can be chemically attached to the ligand and (2) a coupling step, in which the affinity ligand is attached to the activated support. With some techniques, a third step, in which remaining activated groups are removed, is also required. The methods listed in Table 13.3 can be performed either in-house or can be used in the form of preactivated supports available from commercial suppliers (see list in Table 13.2) [25,36]. 

Source Data compiled from Hermanson, G.T. et al., Immobilized Affinity Ligand Techniques, Academic Press, New York, 1992 Kim, H.S. and Hage, D.S., Immobilization methods for affinity chromatography, in Hage, D.S. (ed.). Handbook of Affinity Chromatography, 2nd edn., CRC Press, Boca Raton, EL, 2005, Chap. 3. 

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