Wagner O F, Bergmann I, Binder B R
Anal Biochem. 1985 Nov 15;151(1):7-12. doi: 10.1016/0003-2697(85)90044-2.
A chromogenic substrate autography is described which allows characterization and quantification of serine proteases in crude systems after sodium dodecyl sulfate-polyacrylamide gel electrophoresis or isoelectric focusing. Separation of samples containing proteases by either method is followed by an overlay of the gels on an indicator film prepared by incorporation of a suitable paranitroanilide substrate into agarose. Positions of the proteases are revealed by the formation of yellow-colored zones which can be quantified by densitometry at 405 nm. The technique proved suitable for determination of molecular weights, isoelectric points, and quantitative measurements of amidolytic activities of urokinase, tissue plasminogen activator, tissue and plasma kallikrein, and thrombin in biological fluids and purified preparations.
本文描述了一种显色底物自显影技术，该技术可在十二烷基硫酸钠-聚丙烯酰胺凝胶电泳或等电聚焦后，对粗体系中的丝氨酸蛋白酶进行表征和定量。通过上述任何一种方法分离含蛋白酶的样品后，将凝胶覆盖在通过将合适的对硝基苯胺底物掺入琼脂糖而制备的指示膜上。蛋白酶的位置通过黄色区域的形成来显示，这些黄色区域可通过在405nm处进行光密度测定来定量。该技术被证明适用于测定生物流体和纯化制剂中尿激酶、组织型纤溶酶原激活剂、组织和血浆激肽释放酶以及凝血酶的分子量、等电点和酰胺水解活性的定量测量。
