4.9. Gelatin Zymography

Huh7 cells (3.5 × 10⁶ cells/well) were seeded in 6-well culture plates and incubated in serum-free medium in the presence of the indicated concentrations of GCB for 24 h. The collected medium was mixed with 5× sample buffer (2.5 mM Tris–Cl (pH 6.8), 5% sodium dodecyl sulfate (SDS), 50% glycerol, and 0.05% bromophenol blue) and separated by 10% SDS–polyacrylamide gel electrophoresis (PAGE) with 0.1% gelatin (G1393-20ML, Sigma-Aldrich, St. Louis, MO, USA). The gels were washed twice with wash buffer (50 mM Tris–HCl (pH 7.5), 0.2 M NaCl, 5 mM CaCl2, 0.1 µM ZnCl, and 2.5% Triton X-100) for 30 min at RT and then incubated in substrate buffer (50 mM Tris–HCl (pH 7.5), 0.2 M NaCl, 5 mM CaCl₂, 0.1 µM ZnCl₂, 0.016% NaN₃, and 1% Triton X-100) at 37 °C for 24 h. The gels were fixed with a fixing solution (40% methanol and 10% acetic acid) for 30 min. The gels were then stained with a staining solution (0.25% Coomassie brilliant blue R-250, 45% methanol, and 10% acetic acid) for 1 h, followed by destaining with a destaining solution (5% methanol and 8% acetic acid).