Detection of proteinases in electrophoretograms of complex mixtures

A simple, sensitive technique for detecting proteolytic enzyme zones on electrophoretograms by making contact print zymograms is described. The method is applicable to electrophoretograms prepared on a variety of support media, immunoelectrophoretograms or isoelectric focusing patterns on various media. The contact print zymograms are prepared by placing unfixed, unstained electrophoretograms in contact with a thin film of casein which has diffused into a layer of agarose supported by a hydrophilic polyester film. After staining the casein film with Coomassie blue, the proteolytic zones are detected as clear zones against a blue background. The method can detect as little as 9 ng of trypsin. The utility of the method is illustrated by detection of the proteinase enzymes in thermophilic actinomycete antigen preparations separated by polyacrylamide electrophoresis, crossed immunoelectrophoresis and isoelectric focusing on agarose and granulated dextran supports.