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In this procedure, active gelatinases digest gelatin embedded in a polyacrylamide gel. After Coomassie staining, areas of degradation are visible as clear bands against a darkly stained background.
This protocol is optimized for detecting secreted MMP-9 and MMP-2 activity in conditioned media.
Running the gel
Separating gel (7.5 % acrylamide) | Stacking gel | ||
1.5 M Tris pH 8.8 | 2 mL | 0.5 M Tris pH 6.8 | 1.25 mL |
30% acrylamide | 2 mL | 30% acrylamide | 0.67 mL |
H2O | 2 mL | H2O | 3.08 mL |
4 mg/mL gelatin | 2 mL | 10 % SDS | 50 μL |
10% SDS | 80 μL | 10 % APS | 50 μL |
10% APS | 80 μL | TEMED | 10 μL |
TEMED | 10 μL |
Gel washing and staining
5X non-reducing sample buffer (pH 6.8)
Final concentration | For 250 mL |
4% SDS | 10 g |
20% glycerol | 50 mL of 100% |
0.01% bromophenol blue | 0.025 g |
125 mM Tris-HCl | 4.91 g |
H2O | 200 mL |
Final concentration | For 250 mL |
2.5% Triton X-100 | 6.25 mL of 100% |
50 mM Tris HCl | 12.5 mL of 1 M stock |
5 mM CaCl2 | 625 μL of 2 M stock |
1 μM ZnCl2 | 2.5 μL of 0.1 M stock |
H2O | 228 mL + 2 mL 2% NaN3 |
Final concentration | For 250 mL |
1% Triton X-100 | 2.5 mL of 100% |
50 mM Tris-HCl | 12.5 mL of 1 M |
5 mM CaCl2 | 625 μl of 2 M |
1 µM ZnCl2 | 2.5 μl of 0.1 M |
H2O | 233 mL + 2 mL 2% NaN3 |
For 100 mL | |
Methanol | 40 mL |
Acetic acid | 10 mL |
H2O | 50 mL |
Coomassie Blue | 0.5 g |
For 1 L | |
Methanol | 400 mL |
Acetic acid | 100 mL |
H2O | 500 mL |