Zymogram developing buffer

Protein was extracted from isolated abdominal aortas that had been snap frozen in liquid nitrogen and homogenized in a buffer containing 1 M NaCl, 2 M urea, 0.2 mM PMSF, 50 mM Tris (pH 7.4), 0.1% EDTA, 0.1% Brij-35, and protease inhibitors (10 μg/ml aprotinin, 1 mM phenylmethylsulfonyl fluoride, 10 μg/ml leupeptin), as previously described^{41 (link)}. Samples were sonicated on ice and centrifuged, and supernatants were used to quantify protein content. Protein lysate was placed in a nonreducing zymogram buffer (Cat#161–0764, Bio-Rad, Hercules, CA) and applied without boiling to a 10% zymogram gel (#161–1167, Bio-Rad). Gels were incubated in 2% Triton X-100 at room temperature for 30 min and then rinsed in H₂O for 5 min. Gels were incubated overnight at 37°C with gentle agitation in Zymogram developing buffer (Cat# 161–0766, Bio-Rad) containing 50 mM Tris-HCl, pH 7.5, 200 mM NaCl, 5 mM CaCl2, 0.02% Brij-35. Proteins were stained with Coomassie brilliant blue R-250 solution (Cat# 161–0436, Bio-Rad) and destained with a solution containing 40% methanol, 10% acetic acid, and 50% H₂O.

Mouse recombinant osteopontin protein (mrOPN, No. 441-OP (R&D Systems, Minneapolis, USA); accession number Q547B5, clone F630228I12) was incubated with active MMP-9 (Calbiochem, Billerica, Massachusetts, USA) or activated proMMP-2, -3, or -7 (all from R&D Systems) at a ratio of 3:1 (w/w) in 1× zymogram developing buffer (Bio-Rad, Hercules, California, USA). Pro-form MMPs were activated with 1 mmol/L p-aminophenylmercuric acetate (APMA). All reactions were incubated at 37 °C for 24 h The mixture was dried in the SpeedVac and resuspended in 20 μL 1 mol/L urea– 50 mmol/L ammonium with 10 mmol/L Tris-(2-carboxyethyl)-phosphine (TCEP), followed by 30 min reduction at room temperature. Iodoacetamide (16 mmol/L) was added for alkylation during 30 min at room temperature, in the dark. Sequencing grade modified trypsin (Promega, Madison, Wisconsin, USA) was added at 1:30 (enzyme:protein) and the incubation continued at 37 °C for 18 h. The digests were cleaned using C18 ziptip (Millipore, Billerica, Mass.), followed by analysis using liquid chromatography – tandem mass spectrometry (LC-MS/MS). mrOPN directly digested with trypsin was included as a control and underwent the same procedure.