Sharma Kanika, Bhattacharyya Debasish
Division of Structural Biology and Bioinformatics, CSIR-Indian Institute of Chemical Biology, 4 Raja S.C. Mullick Road, Jadavpur, Kolkata, 7000032, India.
Methods Mol Biol. 2017;1626:125-132. doi: 10.1007/978-1-4939-7111-4_11.
Reverse zymography is a technique by which protease inhibitor(s) in a sample could be electrophoretically separated in a substrate-impregnated acrylamide gel and their relative abundance could be semi-quantified. The gel after electrophoresis is incubated with a protease when the impregnated substrate and all other proteins of the sample are degraded into small peptides except the inhibitor(s) that show clear bands against a white background. Since reverse zymography cannot distinguish between a protease inhibitor and a protein that is resistant against proteolysis, the results should be confirmed from inhibition of protease activity by solution state assay.
反向酶谱法是一种技术，通过该技术可以在浸渍有底物的丙烯酰胺凝胶中对样品中的蛋白酶抑制剂进行电泳分离，并对其相对丰度进行半定量。电泳后的凝胶与蛋白酶一起孵育，此时浸渍的底物和样品中的所有其他蛋白质都被降解成小肽，除了抑制剂，抑制剂在白色背景下显示出清晰的条带。由于反向酶谱法无法区分蛋白酶抑制剂和抗蛋白水解的蛋白质，因此应通过溶液状态分析对蛋白酶活性的抑制来确认结果。
