Economical use of substrates in enzyme activity blots

摘要

A method for the analysis of individual proteinases in crude tissue extracts, which is highly sensitive and uses conservative amounts of substrate, is described. A proteinase activity blot assay was first described by Ohlsson et al.,in which para-nitroanilide (pNA) substrates were incubated with enzymes previously separated by agarose gel electrophoresis and transferred to nitrocellulose. The procedure has now been modified by the use of sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (PAGE) and improved by the incorporation of a more economical blotting system (EconoBlot, LabLogix, Belmont, CA) or a sectored screening device (Mini-Protean II Multi Screen, BioRad, Hercules, CA) to reduce the amount of substrate required. With these modifications, a mixture of enzymes can be economically and quickly evaluated for their ability to hydrolyze substrates diagnostic of certain enzyme classes.