Sds polyacrylamide gel electrophoresis

The activity of MMP-2 and MMP-9 treated with various concentrations of EGCG was evaluated using gelatin zymography. Briefly, following treatment with EGCG in serum-free RPMI-1640 medium for 24 h, the conditioned medium was obtained and the supernatant collected by centrifugation at 4°C and 447.2 × g for 10 min. The samples were loaded and separated by electrophoresis on 10% sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (Sigma-Aldrich) with 1 mg/ml gelatin at 100 V for 2 h at 4°C. Following this, the gels were washed twice in 2.5% Triton X-100 (Sigma-Aldrich) for 30 min at room temperature to remove SDS, and incubated overnight in zymography developing buffer containing 50 mM Tris-HCl and 10 mM CaCl₂ (pH 7.5; Sigma-Aldrich) at 37°C. Subsequent to incubation, gels were stained with 0.5% Coomassie Blue for 30 min at room temperature and stained with 30% methanol and 10% glacial acetic acid (all Beijing Chemical Works, Beijing, China). The gelatinase activity of MMP-2 and MMP-9 was visualized as clear bands against the dark blue background, and band density was measured using Quantity One 4.6.3 software (Bio-Rad Laboratories, Inc.). Results were expressed by the percentage of the density to the control bands. A minimum of three independent experiments were conducted with individual protein samples.