Sodiumdodecyl sulphate-polyacrylamide Gel Electrophoresis (SDS–PAGE)was carried out for the control of the purity and determinationof 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, pH6.8, containing 2% SDS, 10% (v/v) glycerol, 5% ß-mercaptoethanol and 0.002% bromophenol blue). Thesamples were heated at 100?Cfor 10 min before loading in the gel.
The samples (15 µl) were loaded onto the stacking gel andsubjected to electrophoresis at a constant currentof 15 mA using a vertical electrophoresissystem (Vertical Electrophoresis System,Bio-Rad Laboratories, Inc.). After electrophoresis, the gels were stained with 0.
1% Coomassie brilliant blue G-250in 35% methanol and 7% acetic acid anddestained with 7% acetic acid and 35% methanol. The molecular weight of the purified trypsin was estimated bycomparing its Rf with those of protein standards in ladder.Native-PAGEwas performed using 15% separating gel in a similar manner, except that thesample was not heated and SDS and b-mercaptoethanol were omitted from all geland buffer solutions.Casein zymography was performed on Native-PAGE according to themethod of Garciacarreno, Dimes, and Haard (1993) Sampleswere mixed with electrophoresis loadingbuffer 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 substrateto 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.