Immobilization of Wheat Germ Agglutinin

1 M N-acetyl-glucosamine (GlcNAc, 221.2), IM NaCl, 0.1 M borate buffer, pH 8.3 

Dissolve in a centrifugation tube 10 mg wheat germ agglutinin (WGA) in 5 ml of Soln. A. Add 5 g of wet, ice-cold activated gel (from BrCN activation cf. Protocol 3.6.1) and agitate the mixture at a roller shaker for 2 h at RT or overnight at 4 °C. 

After incubation, add 10 ml Soln. B and block at RT further 2 h. Wash the gel consecutively with 50 ml each of Soln. C and Soln. D and repeat this step. Finish coupling by washing on a filter funnel with 100 ml of Soln. E, suck off the buffer and resuspend then in 5 ml of Soln. E. Store the gel in a refrigerator. 

The coupling yield is about 60%. If necessary, check the binding capacity by adsorption of fetuin and elution with 0.1 M N-acetyl-glucosamine in PBS. 

The immobilization of any other protein to cyanogen bromide-activated or N-hydroxysuccinimide- (NHS) activated gels is done the same way. 

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