Gelatinase zymography

Substrate-specific zymography for determination of gelatinolytic activity of MMP-2 and MMP-9 was performed as previously described [48]. Briefly, 20 μg of each protein extract were subjected to gel electrophoresis using 10 % Zymogram (Gelatin) pre cast Gel and zymogram gel was developed according to manufacturer protocol (Bio-Rad Laboratories, Milan, Italy). After electrophoresis, the gel was washed twice 2.3 % triton X-100 for 30 min and incubated in development buffer (50 mM tris, 200 mM NaCl, 5 mM CaCl₂, 0.02 % Brij-35 at 37 °C for 20 h. Gel was then stained with 0.5 % coomassie blue R-250 in staining solution (40 % methanol, 10 % acetic acid, 0.5 % coomassie blue, 100 ml deionized water) at room temperature (RT) for 1 h and was de-stained in destaining solution (40 % methanol, 10 % acetic acid, 250 ml deionized water), until clear lysis bands appeared. To quantify the intensities of the degradated bands, zymogram gel was scanned using ChemiDoc gel scanner (Bio-Rad Laboratories).