Zymography

Samples are prepared in a standard, non-reducing loading buffer for SDS-PAGE. No reducing agent or boiling are necessary since these would interfere with refolding of the enzyme. A suitable substrate (e.g. gelatin or casein for protease detection) is embedded in the resolving gel during preparation of the acrylamide gel. 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 optimized 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. ==Variations on the standard protocol==

The standard protocol may require modifications depending on the sample enzyme; for instance, D. melanogaster digestive glycosidases generally survive reducing conditions (i.e. the presence of 2-mercaptoethanol or DTT), and to an extent, heating. Indeed, the separations following heating to 50 °C tend to exhibit a substantial increase in band resolution, without appreciable loss of activity. A common protocol used in the past for zymography of α-amylase activity was the so-called starch film protocol of W.W. Doane. Here a native PAGE gel was run to separate the proteins in a homogenate. Subsequently, a thin gel with starch dissolved (or more properly, suspended) in it was overlaid for a period of time on top of the original gel. The starch was then stained with Lugol's iodine. Gel zymography is often used for the detection and analysis of enzymes produced by microorganisms. This has led to variations on the standard protocol e.g. mixed-substrate zymography. Reverse zymography copolymerizes 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 visualized as dark bands against a clear (or lightly stained) background. In imprint technique, the enzyme is separated by native gel electrophoresis and the gel is laid on top of a substrate treated agarose. Zymography can also be applied to other types of enzymes, including xylanases, lipases and chitinases. ==See also==