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Gelatin zymography

JR Judd C. Rice
BW Benjamin H. Weekley
TK Tomas Kanholm
ZC Zhihui Chen
SL Sunyoung Lee
DF Daniel J. Fernandez
RA Rachel Abrahamson
AC Alessandra Castaldi
ZB Zea Borok
BD Brian D. Dynlacht
WA Woojin An
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A 4–10% SDS-PAGE gel was cast with 0.1% gelatin in the resolving portion of the gel (10% polyacrylamide, 1 mg/ml gelatin, 375 mM Tris–HCl pH 8.8, 0.1% SDS). The gel lanes were loaded with equivalent amounts of nuclear soluble extracts in 1 × non-denaturing load dye (66 mM Tris–HCl pH 6.8, 10% glycerol, 2% SDS, 0.01% bromophenol blue) and fractionated via electrophoresis. The gel was incubated in 100 ml renaturing solution (50 mM Tris–HCl pH 7.5, 5 mM CaCl2, 2.5% Triton X-100) two times for 30 min to remove SDS, washed in 100 ml diH2O two times for 15 min, and incubated in 100 ml developing solution (50 mM Tris pH 7.5, 10 mM CaCl2, 0.02% NaN3) for 1 h. The developing solution was replaced and the gel was incubated at 37 °C for 18 h. The gel was incubated with 100 ml of staining solution (5% methanol, 10% acetic acid, 0.125% coomassie R-250) for 4 h and then 100 ml of destaining solution (10% MeOH, 5% acetic acid) prior to imaging using a LI-COR Odyssey.

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