Activation of Enzymes with NHS Ester—Maleimide Cross-linkers

Activation of Enzymes with NHS Ester-Maleimide Cross-linkers 

One note should be mentioned before proceeding when conjugating antibody molecules with (3-galactosidase, the antibody usually is activated with sulfo-SMCC first to take advantage of the indigenous sulfhydryl groups on the enzyme. Therefore, if (3-gal is being used, substitute the antibody for the enzyme mentioned in this protocol. 

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 packed and equilibrated prior to starting the modification reaction. Enzymes preactivated with sulfo-SMCC are available from Pierce. 

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