Gelatin zymography for detecting MMP-2 and MMP-9 activity

Supernatant was collected and cultured. After mixing with buffer solution at 1:1, 8 % SDS-PAGE containing 1 % gelatin was added, and electrophoresis was performed for 2 h. The gel was washed with 2.5 % Triton X-100 twice at room temperature for 30 min and transferred to substrate buffer (10 mmol/L Tris Base, 40 mmol/L Tris-Cl, 0.2 mol/L NaCl, 5 mmol/L CaCl₂, 0.02 % Brij 35, pH 7.6). After reaction overnight at 37 °C and fixation for 2 h, Coomassie blue staining was performed for 20 min. Eluent destaining was performed until clear digestion bands were observed. Image analysis was performed on the electrophoresis results.