Human mmp 2

Similarly prepared protein samples as for Western blot were subjected to gelatin zymography. Approximately 50 μg of each sample was loaded per lane into the well of precast gels (10% polyacrylamide minigels containing 0.1% gelatin; Invetrogen) with SDS running buffer (1:1; Novex). Electrophoresis was performed with a Tris-glycine running buffer at 125 V constant voltage for 1.5–2 h. The gel was removed and incubated for 1 hour at room temperature in 100 ml of 2.7% Triton X-100 (renature buffer, Invetrogen) on a rotary shaker, then washed with distilled water 10 times. Each gel was incubated with 100 ml of development buffer (50 mM Tris base, 40 mM HCl, 200 mM NaCl, 5 mM CaCl2, and 0.2% Brij 35; Invetrogen) at 37°C for 14 hours on a rotary shaker. Staining was performed with 100 ml of 0.5% Coomassie blue G-250 in 30% methanol and 10% acetic acid for at least 1 hour, and gels were then destained with three changes of solutions. Gelatinolytic activity was demonstrated as clear zones or bands at the appropriate molecular weights. Human MMP-9 and human MMP-2 (from Chemicon Temecula, CA) were used as standards. The activity of MMP-2 and MMP-9 was quantified in ImageJ software.