Gelatin Zymography for MMP-2 and MMP-9 Analysis

Vein protein extracts (with no dithiothreitol) were run electrophoretically on 8% SDS-polyacrylamide gel supplemented with gelatin (0.1%, Sigma, St. Louis, MO). The gel was then placed in renaturing buffer supplemented with Triton-X-100 (2.5%, Sigma) and gently agitated at room temperature for 30min. The gel was transferred to developing buffer composed of Tris (50mM), NaCl (0.2M), CaCl₂ (5mM), Brij35 (0.02%, Fisher, Pittsburgh, PA), and ZnCl₂ (1μM, Sigma) at an adjusted pH 6.7, first at room temperature for 30min then at 37°C for 16h. The gel was stained with coomassie blue R-250 (0.5%, Sigma) for 30min, then destained in a solution composed of methanol:acetic acid:water at 50:10:40 ratio. Proteolytic areas representing MMP-2 and MMP-9 showed as clear bands against blue background. Actin showed as dark blue area at 43kDa against light blue background. In all experiments, equal amount (1μg) of protein from various tissue samples was used to load the gels. The clear MMP proteolytic bands were analyzed using optical densitometry and ImageJ (NIH), and the integrated gelatinolytic activity was presented as pixel intensity×mm² relative to actin to correct for any differences in sample loading and variations among different gels.^{28 (link)–30 (link)}