SUBSTRATE GEL-ELECTROPHORESIS FOR COMPOSITION AND MOLECULAR-WEIGHT OF PROTEINASES OF PROTEINACEOUS PROTEINASE-INHIBITORS

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. © 1994 Academic Press, Inc. All rights reserved.