Chromogenic substrate autography: a method for detection, characterization, and quantitative measurement of serine proteases after sodium dodecyl sulfate-polyacrylamide gel electrophoresis or isoelectric focusing in polyacrylamide gels |
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| Authors: | O F Wagner I Bergmann B R Binder |
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| Affiliation: | 1. School of Food Engineering, Department of Food and Nutrition, University of Campinas, R. Monteiro Lobato, 80 - Cidade Universitária, Campinas, SP 13083-852, Brazil;2. Department of Management/MAPP, Aarhus University, Fuglesangs Allé 4, 8210 Aarhus V, Denmark;3. Marketing and Consumer Behaviour Group, Wageningen University and Research. Hollandseweg 1, 6706, KN, Wageningen, The Netherlands.;4. XOC editorial, Calderón de la Barca 359, Mexico City 11559, Mexico;1. School of Traditional Chinese Materia Medica, Key Laboratory of Structure-Based Drug Design and Discovery, Ministry of Education, Shenyang Pharmaceutical University, Shenyang 110016, People''s Republic of China;2. School of Pharmaceutical Engineering, Shenyang Pharmaceutical University, Shenyang 110016, People''s Republic of China;3. Chinese People''s Liberation Army 210 Hospital, Dalian 116021, People''s Republic of China |
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| Abstract: | 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. |
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