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Author:
Date:
2013-08-20
[Abstract] This protocol describes a gel-based procedure to detect protease inhibitor activity. In this method gelatin is used as a substrate for proteolysis and is copolymerized within the polyacrylamide matrix. Protein extracts are fractioned by SDS-PAGE and then the gel is treated with the protease of interest, which degrades gelatin, except in the areas where inhibitory activity is present. Inhibition of protease activity appears as colored bands against a clear background after staining with Coomassie Brilliant Blue (Figure 1). The effectiveness of the assay is dependent on the capacity of the protease inhibitor to refold after SDS-PAGE fractionation. Alternatively, it can be performed using native (PAGE) gels. Although the protocol presented here has been standardized to test for subtilisin ...
[摘要] 该协议描述了基于凝胶的程序来检测蛋白酶抑制剂活性。 在该方法中，明胶用作蛋白水解的底物并在聚丙烯酰胺基质内共聚。 通过SDS-PAGE分级分离蛋白质提取物，然后用目标蛋白酶处理凝胶，除了存在抑制活性的区域外，其降解明胶。 在用考马斯亮蓝染色后，蛋白酶活性的抑制表现为针对透明背景的有色条带（图1）。 测定的有效性取决于蛋白酶抑制剂在SDS-PAGE分离后再折叠的能力。 或者，其可以使用天然（PAGE）凝胶进行。 虽然这里提出的协议已经标准化以测试枯草杆菌蛋白酶抑制活性，它可以很容易地适应测试其他蛋白酶和蛋白酶抑制剂。
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