Reverse fibrin autography: a method to detect and partially characterize protease inhibitors after sodium dodecyl sulfate--polyacrylamide gel electrophoresis.
Reverse fibrin autography: a method to detect and partially characterize protease inhibitors after sodium dodecyl sulfate--polyacrylamide gel electrophoresis.
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反向纤维蛋白自显影：十二烷基硫酸钠-聚丙烯酰胺凝胶电泳后检测和部分表征蛋白酶抑制剂的方法。
DOI：
10.1016/0003-2697(84)90113-1
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发表时间：
1984
影响因子：
2.9
通讯作者：
Loskutoff,DJ
中科院分区：
文献类型：
--
作者：
Erickson,LA;Lawrence,DA;Loskutoff,DJ
开发了一种新的技术，即反向纤维蛋白自显影，用于检测在十二烷基硫酸钠存在下，通过聚丙烯酰胺凝胶电泳法分离得到的蛋白酶抑制物。外源蛋白酶被掺入纤维蛋白-琼脂指示剂膜中，最终导致纤维蛋白溶解。当含有抑制剂的丙烯酰胺凝胶被放置在这样的指示剂上时，抑制剂的位置通过在原本透明的纤维蛋白膜中形成不透明的、抗溶解的区域来揭示。这项技术是多才多艺的，因为揭示了各种抑制剂，并半定量，因为指示剂中的裂解阻滞区的大小与接受电泳法的抑制剂的量成比例增加。这种方法不仅可以用来检测具有不同蛋白酶特异性的抑制物，而且还可以区分针对尿激酶或组织型纤溶酶原激活物的抗体的抑制活性。因此，反向纤维蛋白自显影为快速筛选和部分表征复杂生物样品中存在的抑制剂提供了一种方便的新方法。
A new technique, reverse fibrin autography, was developed to detect protease inhibitors previously fractionated by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Exogenous proteases were incorporated into fibrin-agar indicator films, eventually causing the fibrin to lyse. When an acrylamide gel containing inhibitors was placed on top of such an indicator, the positions of the inhibitors were revealed by the formation of opaque, lysis-resistant zones in the otherwise cleared fibrin film. The technique was versatile in that a variety of inhibitors were revealed, and semiquantitative since the size of the lysis-resistant zone in the indicator increased in proportion to the amount of inhibitor subjected to electrophoresis. This approach could be used not only to detect inhibitors having different protease specificities, but also to distinguish between the inhibitor activities of antibodies directed against urokinase or tissue-type plasminogen activator. Thus, reverse fibrin autography offers a convenient new approach to rapidly screen and partially characterize inhibitors present in complex biological samples.