Activation of Enzymes with NHS Ester-Maleimide Crosslinkers

Activation of Enzymes with NHS Ester-Maleimide Crosslinkers 

SMCC-activated enzyme containing sulfhydryl-reactive maleimide groups 

The following protocol describes the activation of HRP with sulfo-SMCC. Activation of other enzymes is done similarly, with the appropriate adjustments in the mass of enzyme added to the reaction to account for differences in molecular weight. 

The gel filtration column described in step 3 should be prepared and equilibrated prior to starting the modification reaction. Enzymes preactivated with sulfo-SMCC are available from Thermo Fisher. 

Dissolve 18 mg of HRP in 0.1M sodium phosphate, 0.15 M NaCl, pH 7.2, at a concentration of 20-30mg/ml. The more highly concentrated the enzyme solution, the more efficient will be the modification reaction. For conjugating smaller quantities of enzyme and antibody, proportionally decrease the amount of the reagents used, while attempting to maintain the same relative concentrations in solution. 

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