Broad range protein ladder

A complex of extracellular proteins was precipitated with ammonium sulfate (608 g per 1 L culture supernatant). After 48 h incubation at 4 °C, the proteins were centrifuged at 3160× g (4 °C) for 45 min in an LMC-4200R centrifuge (Biosan, Latvia). The precipitate was dissolved in 0.01 M Tris HCl buffer (pH 8.2) and dialyzed against 0.005 M Tris HCl buffer (pH 8.2) for 24 h prior to re-centrifugation under similar conditions. The supernatant was used for isoelectric focusing with the Westerberg method in a pH gradient of ampholines and a 0–40% sucrose density in a 110 mL column at 800 V and 4 °C. After 36 h, the resulting mixture was fractionated in 1.5 mL. For each fraction, different parameters were measured: pH, protein content (spectrophotometrically at 280 nm), and enzymatic activity. The purified enzyme was analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) using a discontinuous buffer system [36 (link)]. A separation gel (15%, w/v) and a stacking gel (5%, w/v) were used. The electrophoretic migration of the keratinolytic enzyme was compared with a pre-stained broad-range protein ladder (Thermo Scientific, USA) to detect the protein’s molecular weight after staining with Coomassie Brilliant Blue R-250.

SDS-PAGE was performed according to the protocol of Laemmli^{69 (link)}. Briefly, the protein was mixed with sample buffer (0.075 M Tris–HCl, pH 6.8, 2% SDS, 10% glycerol, 5% 2-mercaptoethanol and bromophenol blue) and heated in a water bath at 95 °C for 5 min. A broad-range protein ladder (Thermo, Rockford, USA) was employed as the standard to determine the molecular mass of protein. Protein separation was achieved via a 12.5% polyacrylamide gel prepared with resolving buffer (1.5 M Tris–HCl, pH 8.8, 0.4% SDS, 100 ml H₂O), 10% ammonium persulfate and 30% acrylamide solution. Electrophoresis was achieved in running buffer (0.025 M Tris, 0.192 M glycine, pH 8.3, H₂O). Protein bands were visualized by staining the gel with CBB-R250 (1% CBB, 10% acetic acid, 50% methanol, 40% H₂O).