Zymography Techniques for the Profiling of Digestive Protease in a Freshwater Invertebrate Model

Zymography is a sensitive and specific technique that enables the detection and characterization of proteases of low abundance. Here, we describe two zymographic techniques, in-gel and in situ zymography, to discover proteases (20-120 kDa) along the gut of apple snails. Proteases of different molecular weights are separated by electrophoresis in gelatin copolymerized sodium dodecyl sulfate (SDS) polyacrylamide gels and then enzymatic activities revealed by Coomassie Blue negative staining. Protease families can be identified in the presence of specific inhibitors. We also use in situ zymography for localizing proteases in intracellular symbiotic corpuscles that habit in the digestive gland of the gastropod Pomacea canaliculata. Different spatial-temporal scenarios of protease synthesis, secretion, and hydrolysis of dietary proteins may be identified by a combination of in-gel and in situ zymography.