Protein electrophoresis and enzyme activity zymograms

Culture supernatants were analyzed by protein electrophoresis according Laemmli [46]. An aliquot of 21 μl of supernatant from each culture was mixed with 4× NuPAGE LDS sample buffer (Thermo Fisher Scientific), heated at 70°C for 10 min, and subjected to electrophoresis at 120 V for approximately 1 h on a 12.5% (w/v) tris-glycine gel using a running buffer containing 1.4% (w/v) glycine, 0.1% (w/v) SDS and 24 mM Tris. The protein gel was stained overnight in Coomassie Brilliant Blue G-250 (Bio-Rad, CA, USA) and destained in 1% (v/v) acetic acid. To detect protease activity of the secreted proteins, another set of supernatants (not heated) was applied to zymogram gels made of 12.5% (w/v) Tris-glycine gels containing 0.1% (w/v) casein (from bovine milk, Sigma, Australia). Following electrophoresis, zymogram gels were soaked in 2.5% (v/v) Triton-X 100 to for 30 min twice and washed 3 × 10 min in MilliQ water to renaturate proteins and restore enzyme activity. The gels were then incubated for 16 h in 0.03 mol l^-1 Tris-HCl buffer pH 7.5 at 37°C. After incubation, the zymogram gels were subjected to the same staining and destaining procedures as the SDS-PAGE gels [47].