Electrophoretic Zymography of Protease Using Azocasein

Zymography is an electrophoretic technique used to demonstrate enzyme activity during separation in polyacrylamide gels under nonreducing conditions. This kind of analysis can be used in association with electrophoresis (SDS-PAGE) to show which protein is correlated with a given activity. Proteases are the most used enzymes for this technique since the polyacrylamide gel can be impregnated with azocasein, a casein derivative with an added sulfanilamide group (orange color). Furthermore, azocasein can be degraded while the protease is separated through the gel. The degradation is easily revealed by dyeing the azocasein with Coomassie blue, with the nonblue area in the gel being the zone where the separated protease shows activity. Zymography has been widely used for the identification and characterization of new proteolytic enzymes, but it can detect latent and active forms of enzymes in cells, tissues, or biological fluids.