Gelatin zymography

To determine the activity of matrix metalloproteinase-2 (MMP-2) and matrix metalloproteinase-9 (MMP-9) in the conditioned media, MDA-MB-231 cells (2 × 10⁵/ well) were seeded in 6-well plates. After treatment with DMSO or 75, 100, 150, 200 or 250 µg /ml of extract for 24 hours, the supernatants were collected and subjected to gel electrophoresis on 10% running gels containing 0.1% gelatin. The gels were washed in 2.5% Triton X-100 for 30 min, followed by incubation for 20 hours at 37℃ in the incubation buffer containing 50 mM Tris-HCl (pH 7.5), 0.2 M NaCl, 1 mM CaCl₂ and 0.2% NaN₃. The gels were stained in 0.5% Coomassie brilliant blue (Sigma, St. Louis, USA) then destained with destaining solution (45% methanol and 10% acetic acid) and photographed.