dodecyl sulphate-polyacrylamide Gel Electrophoresis (SDS–PAGE)
was carried out for the control of the purity and determination
of molecular weight of the purified trypsin according to the method of Laemmli (1970) using 5% (w/v) stacking and 15% (w/v) separating gels.
Protein solutions were mixed at 2:1 (v/v) ratio with the SDS–PAGE sample buffer (0.0625 M Tris-HCl, pH
6.8, containing 2% SDS, 10% (v/v) glycerol, 5% ß-mercaptoethanol and 0.002% bromophenol blue). The
samples were heated at 100?C
for 10 min before loading in the gel. The samples (15 µl) were loaded onto the stacking gel and
subjected to electrophoresis at a constant current
of 15 mA using a vertical electrophoresis
system (Vertical Electrophoresis System,
Bio-Rad Laboratories, Inc.). After electrophoresis, the gels were stained with 0.1% Coomassie brilliant blue G-250
in 35% methanol and 7% acetic acid and
destained with 7% acetic acid and 35% methanol. The molecular weight of the purified trypsin was estimated by
comparing its Rf with those of protein standards in ladder.
was performed using 15% separating gel in a similar manner, except that the
sample was not heated and SDS and b-mercaptoethanol were omitted from all gel
and buffer solutions.
Casein zymography was performed on Native-PAGE according to the
method of Garciacarreno, Dimes, and Haard (1993) Samples
were mixed with electrophoresis loading
buffer and electrophoresed on a native PAGE. After electrophoresis, the gel was submerged in 100 ml of 1% (w/v) casein in 100 mM Tris–HCl buffer, pH 8.0 for 60 min at 4?C to allow the substrate
to penetrate into the gel and then incubated for 20 min at 60?C for the development of enzyme activity bands. After washing, the gel was stained with Coomassie Brilliant Blue R-250. Development of a clear zone on the blue background of the gel indicated the presence of protease activity.