Coupling to Cyanogen Bromide-Activated Gels

To immobilize a protein the biologic activity of which depends on a cofactor, this cofactor has to be present in sufficient concentration during the immobilization step. Thus, for example, the lectin Concanavalin A is immobilized in the presence of a buffer containing 0.1 M glucose or a-methylmannoside, 1 mM calcium chloride and 1 mM manganese chloride. 

Important The active center of a protein and the binding site to its ligate has to be protected by substrate, cofactor, ligand, or their analogue during coupling to the chromatographic support. 

5 M NaCl in 0.1 M borate, Tricine, TAPS, or carbonate buffer, pH 9.5 

Wash freshly activated gel (see Protocol 3.6.1) with huge amounts of ice-cold water or in the case of commercially available activated gels, e.g., BrCN-activated Sepharose 6B, with ice-cold 0.001 N HCl. 

After the second incubation wash the gel alternating with the 50-fold volume of Soln. A and B, at least twice. Bring the gel to neutral pH by rinsing with Soln. C. 

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