This protocol describes a gel-based procedure to detect protease inhibitor activity. In this method gelatin is used as a substrate for proteolysis and is copolymerized within the polyacrylamide matrix. Protein extracts are fractioned by SDS-PAGE and then the gel is treated with the protease of interest, which degrades gelatin, except in the areas where inhibitory activity is present. Inhibition of protease activity appears as colored bands against a clear background after staining with Coomassie Brilliant Blue (Figure 1). The effectiveness of the assay is dependent on the capacity of the protease inhibitor to refold after SDS-PAGE fractionation. Alternatively, it can be performed using native (PAGE) gels. Although the protocol presented here has been standardized to test for subtilisin inhibitory activity, it can easily be adapted to test other proteases and protease inhibitors.


Figure 1. Protease inhibitor activity by zymogram analysis of different purified fractions and a protein crude extract. Zymogram was performed after SDS-PAGE. One microgram of protein was loaded to each lane. CE: Crude extract; QS: Fraction from anion exchange chromatography (Q-sepharose); Fractions 35-36: Obtained after a size exclusion chromatography (Superdex 200). M: Molecular markers. Arrows indicate inhibition activity.