Gelatin Zymography
Cell lysis samples (20 ug of protein per sample) were prepared with 4x Laemmli buffer without 2-mercaptoethanol and samples were not boiled. Samples were electrophoresed through 8% tris-glycine polyacrylamide gel with 0.1% gelatin and gels were washed with 2.5% Triton X-100 3 times for 20 minutes each. Gels were incubated in an incubation buffer (NaCl, CaCl2, Tris base) overnight at 37oC. The next day, the gel was stained with 0.05% Coomassie Brilliant Blue G-250 stain (Sigma B1131) for 1 hour and destained with a destaining solution (methanol, glacial acetic acid, ddH2O) overnight at 25oC. Bands were visualized (Azure Biosystems).