Gelatin Zymography for MMP-2 Activity

Gelatin zymography was used to evaluate the MMP-2 activity in the cardiac tissue. The protein concentration was determined by the Bradford method, and then, 70 μg protein was loaded to the 8% sodium dodecyl sulfate- (SDS-) polyacrylamide gel copolymerized with gelatin (20 mg/mL; type A from porcine skin; Sigma). Following electrophoresis, the gels were washed with 2.5% Triton X-100 and incubated overnight at 37°C in an incubation buffer containing 50 mM Tris-HCl, 150 mM NaCl, and 5 mM CaCl₂. A staining method was performed by using 0.05% Coomassie Brilliant Blue, and then, a mixture of 4% methanol and 8% acetic acid was added to the gels. Following washing procedures, the gels were digitally scanned and analyzed by using Quantity One software. MMP-2 human recombinant (Sigma-Aldrich, Hungary) was used as a positive control to identify MMP-2 activities, which were expressed as intensity × mm² [16 (link)].