A biotinylated catalase was prepared by adding a fixed amount (2 mg/ml) of catalase solution (phosphate buffer 100 mM pH 6.0) to a 100 times molar excess of biotinamidocaproate N-hydroxysuccinimide ester. The biotiny-lated reaction was carried out in a vial under constant stirring at room temperature for 3h. At this time, 10 mg of glycine was added to react with the unused biotin. Removal of all small-molecular-weight reactants and products was achieved by chromatography through a Sephadex G 25 column from SIGMA (USA) (molecular weight cut off of 10,000).
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