Quantitative measurement of protease activities in slab polyacrylamide gel electrophoretograms

A simple, rapid, and inexpensive method for the quantitative measurement of protease activity in slab polyacrylamide gel electrophoretograms is described. An electrophoretogram containing separated test samples and dilutions of a standard protease was placed in contact with a thin (0.7-mm) agar gel slab containing gelatin substrate. After 30 min incubation at 37°C, the unhydrolyzed gelatin was precipitated with saturated ammonium sulfate for 10 min. The developed substrate slab proteolytic zymogram was placed between two clear plastic sheets for analysis by densitometry at 525 nm. A standard graph of peak height in the densitometric scan against quantity of standard protease in micrograms or in proteolytic units was plotted. When measurements of a test sample were made in six independent experiments from the initial linear part of the standard curves the mean ± standard deviation was 3.2 ± 0.2 proteolytic units.