Gelatin zymography protocol
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Protocol for using gelatin zymography to detect MMP activity in conditioned media.
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.
Preparation of conditioned media
- Plate cells cultured in fetal bovine serum (FBS) in a six-well plate (2 mL/well) or in a 75 cm2 flask (10 mL).
- At 70–80% confluency, remove FBS media. Wash cells twice with FBS-free media and continue to grow cells in FBS-free media.
The duration of growth in FBS-free media must be optimized for the cell line: for example, 231G and 468 breast cancer cells require a 40–44 h growth period before collection of conditioned media. - Collect conditioned media and centrifuge or filter to eliminate dead cells.
- Concentrate conditioned media 10X.
Gelatin zymography
Running the gel
- Dilute conditioned media so that all samples have the same protein concentration.
For each sample, test one aliquot at a low protein concentration (5 µg/mL) and one at a high protein concentration (15 µg/mL). - Add 5X non-reducing sample buffer to your samples.
- Prepare a 7.5% acrylamide gel containing gelatin. Use a 1 mm thickness plate. See the table below for details.
- Load sample to each well; typically 10 μL protein per well is suitable. Include a protein molecular weight marker in one well.
- Run the gel at 150 V until good band separation is achieved.
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
- Wash the gel 2 x 30 min with washing buffer at room temperature with agitation.
Soaking the gel in washing buffer removes SDS from the gel. - Rinse for 5–10 min in incubation buffer at 37°C with agitation.
- Replace with fresh incubation buffer and incubate for 24 h at 37°C.
The incubation buffer contains cofactors necessary for the gelatinase reaction to occur. - Stain the gel with staining solution for 30 min to 1 h at room temperature with agitation. Rinse with H2O until excess staining solution is removed.
- Incubate with destaining solution until bands can clearly be seen.
Areas of enzyme activity appear as white bands against a dark blue background.
Buffers
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 |
0.5 g |
For 1 L | |
Methanol | 400 mL |
Acetic acid | 100 mL |
H2O | 500 mL |