Reductive amination for solid-phase coupling of protein. A practical alternative to cyanogen bromide.
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.
D000180:Acrylic Resins; D000906:Antibodies / Q000032:analysis; D002846:Chromatography, Affinity / Q000379:methods*; D003488:Cyanogen Bromide / Q000494:pharmacology*; D005169:Factor VIII / Q000302:isolation & purification; D006863:Hydrogen-Ion Concentration; D007074:Immunoglobulin G / Q000032:analysis; D010084:Oxidation-Reduction; D010504:Periodic Acid / Q000494:pharmacology; D011506:Proteins; D012545:Schiff Bases; D013205:Staphylococcal Protein A
D000180:Acrylic Resins; D000906:Antibodies; D007074:Immunoglobulin G; D011506:Proteins; D012545:Schiff Bases; C037239:Sephacryl Superfine; D013205:Staphylococcal Protein A; D010504:Periodic Acid; D005169:Factor VIII; D003488:Cyanogen Bromide