Retaining in-gel zymographic activity of cysteine proteases via a cysteine-supplemented running buffer

Zymography is a powerful technique to separate and identify different enzymatic activities on a standard acrylamide gel. For oxidation prone enzymes such as cysteine proteases however, the oxidizing species generated by electrolysis of the gel running buffer may result in partial or complete inactivation, thus compromising the final readout. This can be only partially remedied by subsequent treatment of the gel with reducing agents. We demonstrate the generation of reactive oxidizing species during electrophoresis and discovered that supplementation of the gel running buffer with a minimum of 5 mM cysteine prevents enzyme inactivation and allows retention of proteolytic activity as measured by zymography on model substrate N α-benzoyl-l-arginine p-nitroanilide, without at the same time altering the mobilities of the gel proteins.