An overlay technique for postelectrophoretic analysis of proteinase spectra in complex mixtures using p-nitroanilide substrates.

A simple method with improved sensitivity for the temperature dependent, and enzyme activity can be lost detection of diVerent groups of proteinases in complex mixtures separated by polyacrylamide electrophoresis and overlaid with a nitrocellulose membrane impregnated with speciWc p-nitroanilide (pNA)1 substrates is described. A drawback of the direct testing of proteinase activity in a polyacrylamide gel with chromogenic peptide substrates is the high degree of diVusion of lowmolecular-mass reaction products. Ohlsson et al. [1] proposed the transfer of proteinases to a nitrocellulose membrane after electrophoresis in a 1% agarose gel with subsequent detection of activity after incubation with pNA substrate solutions. Later this technique was transferred to SDS–PAGE by means of electrotransfer and was actively used for the characterization of digestive proteinases in insects [2,3]. However, conservation of proteinase activity during electrotransfer is time and