Recombinant rat mouse mmp 2 standard

MMP-2 was analyzed accordingly to previous studies [11 (link),32 (link)]. In short, we diluted samples of left ventricular muscle (30 mg) in an extraction buffer (50 mM Tris pH 7.4, 0.2 M NaCl, 0.1% Triton X, and 10 mM CaCl₂); then, we crushed and centrifuged them before quantifying the protein amount in the samples via the Bradford method. Then, we diluted samples with 20 µg of protein in a sample buffer (0.5 M Tris pH 6.8, 50% glycerol, and 0.05% bromophenol blue) and loaded them into wells of 8% SDS-polyacrylamide gel containing 1% gelatin. We performed electrophoresis at 110 V for 2 h in a Bio-Rad mini-protean system in the presence of the running buffer (Tris-Glycine-SDS pH 8.3). Afterward, we washed the gels with Triton X-100 2.5% and Tris-HCl 50 mM pH 8.4 and incubated them for 17 h at 37 °C with continuous agitation. Last, we stained the gel with 2.5% Coomassie brilliant blue followed by a 1 h bath in 30% methanol and 10% acetic acid at room temperature. We photographed the gels and analyzed them by Gel-Pro 3.2 (Media Cybernetics, Rockville, MD, USA). We included the same control sample in each gel to normalize the results. We confirmed the MMP-2 position in the gels by a recombinant rat/mouse MMP-2 standard (R&D System, Minneapolis, MN, USA).

Approximately 30 mg of LV tissue was added to extraction buffer (Tris 50 mM pH 7.4, NaCl 0.2 M, Triton X 0.1%, and CaCl₂ 10 mM), crushed and centrifuged. Protein was quantified in supernatant by the Bradford method [28 (link)]. Samples (10 μg of protein) were diluted in sample buffer (Tris 0.5 M pH 6.8, glycerol 50% and bromophenol blue 0.05%) and submitted to electrophoresis on 8% polyacrylamide plus 1% gelatin gels. After running, the gels were washed with Triton X-100 2.5% and Tris-HCl 50 mM pH 8.4 and incubated for 17 h at 37 °C with continuous agitation (tris-HCl 50 mM pH 8.4, CaCl₂ 500 mM buffer). Subsequently, gels were stained with Coomassie brilliant blue 2.5% and discolored by 30% methanol and 10% acetic acid. A control sample was included in each gel for normalizing results. Matrix metalloproteinase (MMP)-2 position in the gels was confirmed by recombinant rat/mouse MMP-2 standard (R&D Systems, Minneapolis, MN, USA). The gels were photographed by ImageQuant LAS (General Electrics Healthcare, Arlington Heights, IL, USA) and analyzed by Gel-Pro 3.2 (Media Cybernetics, Rockville, MD, USA).