Amplification of chromogenic staining of proteases within electrophoretic gels.
A method is described for amplification of bands generated by chromogenic substrates following their reaction with proteases within electrophoretic gels. Chromogenic substrates, consisting of synthetic peptides containing the chromophore 4-nitroaniline (paranitroaniline, PNA), are applied directly to the surface of agarose or acrylamide gels. Protease activity within the gel results in the enzymatic amidolysis of the chromogenic substrates, releasing free PNA. The yellow PNA chromogen is then derivatized by reaction with p-dimethylaminocinnamaldehyde (DACA) to form a purple Schiff base compound. The resultant complex has a significantly higher molar absorbancy than the original PNA chromogen, thus increasing the sensitivity for low levels of amidolytic activity. The derivatized chromogen is easily visualized for photography.
Roth
R I
RI
Department of Laboratory Medicine, University of California, School of Medicine, San Francisco.
Levin
J
J
D000814:Aniline Compounds; D000818:Animals; D001779:Blood Coagulation Factors / Q000032:analysis; D004587:Electrophoresis, Agar Gel / Q000379:methods; D006737:Horseshoe Crabs; D007202:Indicators and Reagents; D008970:Molecular Weight; D010447:Peptide Hydrolases / Q000032:analysis*
D000814:Aniline Compounds; D001779:Blood Coagulation Factors; D007202:Indicators and Reagents; C019498:4-nitroaniline; D010447:Peptide Hydrolases