Sodium dodecyl sulphate–polyacrylamide gel electrophoresis
(SDS–PAGE)
SDS–PAGE was carried out for the control of the purity and
determination of molecular weight of the purified enzyme, as described
by Laemmli (1970), using 5% (w/v) stacking and 15% (w/
v) separating gels. Samples were prepared by mixing the purified
enzyme at 1:5 (v/v) ratio with the SDS–PAGE sample buffer
(10 mM Tris–HCl buffer, pH 8.0), 2.5% SDS, 10% glycerol, 5% bmercaptoethanol
and 0.002% bromophenol blue). The samples
were heated at 100 C for 5 min before loading in the gel. After
electrophoresis, the gel was stained with 0.25% Coomassie Brilliant
Blue R-250 in 45% ethanol, 10% acetic acid, and destained with 5%
ethanol and 7.5% acetic acid. The molecular weight of the trypsin
was estimated using a low-molecular-weight calibration kit as
Sodium dodecyl sulphate–polyacrylamide gel electrophoresis(SDS–PAGE)SDS–PAGE was carried out for the control of the purity anddetermination of molecular weight of the purified enzyme, as describedby Laemmli (1970), using 5% (w/v) stacking and 15% (w/v) separating gels. Samples were prepared by mixing the purifiedenzyme at 1:5 (v/v) ratio with the SDS–PAGE sample buffer(10 mM Tris–HCl buffer, pH 8.0), 2.5% SDS, 10% glycerol, 5% bmercaptoethanoland 0.002% bromophenol blue). The sampleswere heated at 100 C for 5 min before loading in the gel. Afterelectrophoresis, the gel was stained with 0.25% Coomassie BrilliantBlue R-250 in 45% ethanol, 10% acetic acid, and destained with 5%ethanol and 7.5% acetic acid. The molecular weight of the trypsinwas estimated using a low-molecular-weight calibration kit as
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