Gelatin Zymography for Protease Activity

Fresh brain tissues were immersed in RIPA lysis buffer (P0013K, Beyotime, China) at a ratio of 1:4 (w: v) and centrifuged at 4°C (12,000 rpm, 15 min) after homogenization. The protein concentration was determined by a BCA concentration determination kit (P0010, Beyotime, China). The supernatant and loading buffer (P0016N, Beyotime, China) were mixed at a ratio of 4:1. A total of 120 μg of total protein was loaded into an 8% SDS‒PAGE gel containing 10% gelatin. After electrophoresis at 80 V in ice water for 3 h, each gel was washed in 2.5% Triton X-100 for 30 min 3 times. Then, gels were treated with incubation buffer (50 mM Tris-HCL, 0.2 M NaCl, 5 mM CaCl2, 1 µM ZnCl2, 0.02% Brij-35, pH 7.5) for 48 h at 37°C on a shaking table. Gels were stained with 0.5% Coomassie brilliant blue R250 (ST1123, Beyotime, China) for 3 h and then dyed with 30% methanol containing 10% acetic acid until the emergence of an appropriate color discrepancy. The clear bands on the zymogram represented gelatinase activity.