Zymography
Zymography is an electrophoretic technique, based on SDS-PAGE, that includes a substrate copolymerised with the polyacrylamide gel, for the detection of enzyme activity. Samples are prepared in the standard SDS-PAGE treatment buffer but without boiling, and without a reducing agent. Following electrophoresis, the SDS is removed from the gel (or zymogram) by incubation in unbuffered Triton X-100, followed by incubation in an appropriate digestion buffer, for an optimised length of time at 37°C. The zymogram is subsequently stained (commonly with Amido Black or Coomassie Brilliant Blue), and areas of digestion appear as clear bands against a darkly stained background where the substrate has been degraded by the enzyme. Gelatin is the most commonly used substrate, and is useful for demonstrating the activity of gelatin-degrading proteases, but zymography has been applied to a variety of enzymes, including xylanases, proteases, lipases, etc. Reverse zymography copolymerises both the substrate and the enzyme with the acrylamide, and is useful for the demonstration of enzyme inhibitor activity. Following staining, areas of inhibition are visualised as dark bands against a clear (or lightly stained) background.
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