After electrophoresis, the gel was washed twice for 1 h each with 100 ml of 2.5% Triton X-100 in water to replace SDS and separation buffer in the gel. The incubation of the washed gel at 40◦C for 1 h in 100 mM glycine-NaOHbuffer at pH 10 supplemented with 2 mM CaCl2 produced a casein cleared zone at the location of the proteolytic band.