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).