Detection of proteases by clotting of casein after gel electrophoresis

Clotting of casein provides a sensitive method for detection of proteases after gel electrophoresis. The method is here designated "caseogram". After electrophoresis the gel was equilibrated with 0.15-0.3 m sodium acetate, pH 5.3, and an 1% agarose gel containing 1% skim-milk powder in 0.1 m sodium acetate, pH 5.3, was placed on top of the electrophoresis gel. By incubation at 37°C for 2 h the protease-containing zones produced distinct precipitates in the skim-milk gel. For permanet documentation the skim-milk gel was stained with amido black. The detection limit for pepsin A is 5 ng in the caseogram against 25 ng by hemoglobin digestion at pH 2.5. For calf chymosin it is 1 ng against 100 ng by digestion of hemoglobin at pH 3.5. Caseograms work well after agar gel electrophoresis, after different types of immunoelectrophoresis, and after isoelectric focusing or disc electrophoresis in polyacrylamide gels. Since inert proteins do not interfere with the detection, the method is especially suitable for analysis of crude samples. Samples containing pepsinogen or pepsinogen-like zymogens may be activated at pH 2 before equilibration at pH 5.3.