US EPA

5198314 

Journal Article 

Reductive amination for solid-phase coupling of protein. A practical alternative to cyanogen bromide 

Hornsey, VS; Prowse, CV; Pepper, DS 

1986 

Yes 

Journal of Immunological Methods
ISSN: 0022-1759 

93 

1 

83-88 

English 

3021857 

10.1016/0022-1759(86)90436-9 

WOS:A1986E449800012 

For coupling proteins to Sephacryl gels periodate oxidation of these gels was investigated as an alternative method to cyanogen bromide activation. Optimum conditions were studied with respect to periodate concentration, time of oxidation, pH and type of coupling buffer, concentration of protein, temperature and time of protein uptake, and protein leakage after coupling. The effects of sodium cyanoborohydride and ascorbic acid as reducing agents, and of manganese ions as a potential catalyst were investigated. Using the derived optimum conditions, stable solid-phased antibodies were produced in high yield and used to adsorb factor VIII from plasma. These gels were stable for many weeks, as was the intermediate oxidised gel. Reductive amination for coupling proteins to oxidised Sephacryl gels results in increased binding and lower leakage than is obtained with cyanogen bromide activated agarose.