Zymography is an electrophoretic technique, based on SDS-PAGE, that includes a substrate copolymerized with the polyacrylamide gel, for the detection of enzyme activity. [1] 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 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. These protocols, however, are subject to much adjustment. 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. [2] [3]
Plasmodium knowlesi hemoglobinase imprint.[4]

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, chitinases, etc.

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.[5] The starch was then stained with Lugol's iodine.

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.

Imprint technique: the enzyme is separated by native gel electrophoresis and the gel is laid on top of a substrate treated agarose.[4]

References

1. ^ Lantz MS, Ciborowski P (1994). "Zymographic techniques for detection and characterization of microbial proteases.". Methods Enzymol. 235: 563–594. doi:10.1016/0076-6879(94)35171-6. PMID 8057927.
2. ^ Martínez TF, Alarcón FJ, Díaz-López M, Moyano FJ (2000). "Improved detection of amylase activity by sodium dodecyl sulfate-polyacrylamide gel electrophoresis with copolymerized starch.". Electrophoresis 21 (14): 2940–2943. doi:10.1002/1522-2683(20000801)21:14<2940::AID-ELPS2940>3.0.CO;2-S. PMID 11001307.
3. ^ Snoek-van Beurden PA, Von den Hoff JW (2005). "Zymographic techniques for the analysis of matrix metalloproteinases and their inhibitors". Biotechniques 38 (1): 73–83. doi:10.2144/05381RV01. PMID 15679089.
4. ^ a b Hempelmann, E., Putfarken, B., Rangachari, K., Wilson, R.J.M. (1986). "Immunoprecipitation of malarial acid endopeptidase". Parasitology 92: 305–312. doi:10.1017/S0031182000064076. PMID 3520446.
5. ^ Doane WW (1969). "Amylase variants in Drosophila melanogaster: linkage studies and characterization of enzyme extracts". J Exp Zool 171 (3): 31–41. PMID 5348624.