García-Carreño F L, Dimes L E, Haard N F
Centro de Investigaciones Biológicas de BCS, La Paz, México.
Anal Biochem. 1993 Oct;214(1):65-9. doi: 10.1006/abio.1993.1457.
A rapid, sensitive, and generally applicable substrate-sodium dodecyl sulfate-polyacrylamide gel electrophoresis method for detection of proteinases or proteinaceous protease inhibitors in biological preparations is described. Electrophoretic separation of proteinases or proteinaceous proteinase inhibitors in the sample using sodium dodecyl sulfate-polyacrylamide gel slabs is followed by immersion of the gel in (1) a protein substrate solution for detection of proteinases or (2) an appropriate proteinase solution, and then in a protein substrate solution for detection of proteinase inhibitors. Some advantages of the reported method over previously described techniques that incorporate substrate into the gel matrix are: (1) the development of bioactive bands, as well as staining and washing, is accomplished more quickly, i.e., 4 to 6 h; (2) the trailing of proteolysis which often appears in copolymerized substrate systems is eliminated; (3) the method is applicable to assay at pH values other than those used for electrophoresis; (4) molecular weight markers can be visualized on the same gels; (5) proteinaceous proteinase inhibitors can also be examined in the biological samples; and (6) the sensitivity is several times higher than that of former assays.
本文描述了一种快速、灵敏且普遍适用的底物 - 十二烷基硫酸钠 - 聚丙烯酰胺凝胶电泳法，用于检测生物制剂中的蛋白酶或蛋白质类蛋白酶抑制剂。使用十二烷基硫酸钠 - 聚丙烯酰胺凝胶板对样品中的蛋白酶或蛋白质类蛋白酶抑制剂进行电泳分离后，将凝胶浸入：（1）用于检测蛋白酶的蛋白质底物溶液中，或（2）适当的蛋白酶溶液中，然后再浸入用于检测蛋白酶抑制剂的蛋白质底物溶液中。与先前将底物掺入凝胶基质的技术相比，该方法的一些优点包括：（1）生物活性条带的显影以及染色和洗涤更快完成，即4至6小时；（2）消除了在共聚底物系统中经常出现的蛋白水解拖尾现象；（3）该方法适用于在电泳所用pH值以外的其他pH值下进行测定；（4）分子量标记物可在同一凝胶上可视化；（5）蛋白质类蛋白酶抑制剂也可在生物样品中进行检测；（6）灵敏度比以前的测定方法高几倍。
