Gelatin zymography protocol

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.

View protocol as a PDF

Preparation of conditioned media

  1. Plate cells cultured in fetal bovine serum (FBS) in a six-well plate (2 mL/well) or in a 75 cm2 flask (10 mL).
  2. 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.
  3. Collect conditioned media and centrifuge or filter to eliminate dead cells.
  4. Concentrate conditioned media 10X.

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​Gelatin zymography


​Running the gel

  1. 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).
  2. Add  5X non-reducing sample buffer to your samples.
  3. Prepare a 7.5% acrylamide gel containing gelatin. Use a 1 mm thickness plate. See the table below for details.
  4. Load sample to each well; typically 10 μL protein per well is suitable. Include a protein molecular weight marker in one well.
  5. Run the gel at 150 V until good band separation is achieved.
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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

  1. Wash the gel 2 x 30 min with washing buffer at room temperature with agitation.
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    Soaking the gel in washing buffer removes SDS from the gel.
  2. Rinse for 5–10 min in incubation buffer at 37°C with agitation.
  3. 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.
  4. 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.
  5. Incubate with destaining solution until bands can clearly be seen.

    Areas of enzyme activity appear as white bands against a dark blue background.

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Buffers

5X non-reducing sample buffer (pH 6.8)

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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


Washing buffer (pH 7.5)

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


Incubation buffer (pH 7.5)

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


Staining solution


For 100 mL

Methanol

40 mL

Acetic acid

10 mL

H2O

50 mL

Coomassie Blue

0.5 g

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​Destaining solution


For 1 L

Methanol

400 mL

Acetic acid

100 mL

H2O

500 mL