Gelatin zymography
Posted on Friday, October 15, 2004
Description
This technique is a modification of SDS-PAGE, based on the incorporation of an enzymatic
substrate (gelatin) into the electrophoretic gel and the incubation of the gel in appropriate buffers.
It allows to visualize gelatin-degrading enzymes at picogram level.
Procedure
1 Prepare a gel with a final concentration of acrylamide of 7,5%, containing 1mg/ml of gelatin.
2 Run electrophoresis at 150V constant voltage. Do not boil samples, neither use reducing
agents in sample buffer, in order to preserve enzymatic activity.
3 After electrophoresis, wash the gels in washing buffer for 1 hour at room temperature on an
orbital shaker.
4 Incubate the gels in incubation buffer for18 hours at 37°C.
5 Stain the gels with Coomassie Blue for 30 min. Destain with a methanol/acetic acid solution.
Bands of lytic activity will appear clear on a dark background.
Recipes
Washing Buffer: 2,5% Triton X-100 in 50mM Tris/HCl pH 7.5.
Incubation Buffer: 50mMTris/HCl pH 7.5, 10mM calcium chloride, 0.15 M NaCl,0.02% Sodium
Azide.
To prepare lower gel containing 1 mg/ml gelatin:
Acrylamide/Bis-Acrylamide solution 30:08 3,75ml
1.5M Tris/HCl pH 8.8 3,75ml
Gelatin solution 0,2% 7,3 ml
SDS 10% 150 ul
APS 10% 75 ul
TEMED 7,5 ul
Supplies
All reagents are from Sigma-Aldrich.
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