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Gelatin zymography protocol

Protocol for using gelatin zymography to detect MMP activity in conditioned media.
Last edited Thu 13 Apr 2023

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.

Stage 1 - Preparation of conditioned media

Materials required

  • Your cells
  • Six-well plates or flasks
  • FBS-free media
  • Centrifuge

Steps

1

Plate cells cultured in fetal bovine serum (FBS).

  • We can plate cells 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.
3

Collect conditioned media and centrifuge or filter to eliminate dead cells.

4

Concentrate conditioned media 10X.

Stage 2 - Gelatin zymography: running the gel

Materials required

  • Separating gel (7.5% acrylamide): 2 mL of 1.5 M Tris pH 8.8, 2 mL of 30% acrylamide, 2 mL of H2O, 2 mL of 4 mg/mL gelatin, 80 μL of 10% SDS, 80 μL of 10% APS, 10 μL of TEMED.
  • Stacking gel: 1.25 mL of 0.5 M Tris pH 6.8, 0.67 mL of 30% acrylamide, 3.08 mL of H2O, 50 μL of 10%SDS, 50 μL of 10% APS, 10 μL of TEMED.
  • 5X non-reducing sample buffer (pH 6.8): 4% SDS, 20% glycerol, 0.01% bromophenol blue, 125 mM Tris-HCl, H2O.

Recipe to prepare 250 mL of 5X non-reducing sample buffer:

ReagentWeight/volume for 250 mLFinal concentration
SDS10 g4% 
Glycerol50 mL of 100% glycerol20% 
Bromophenol blue0.025 g0.01%
Tris-HCl
4.91 g125 mM 
H2O200 mL-

Steps

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.
4

Load sample to each well.

  • Typically, 10 μL protein per well is suitable. 
5

Run the gel at 150 V until good band separation is achieved.

Stage 3 - Gel washing and staining

Materials required

  • Washing buffer (pH 7.5)
Final concentrationFor 250 mL
2.5% Triton X-100 6.25 mL of 100%
50 mM Tris-HCl12.5 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
H2O228 mL + 2 mL 2% NaN3
  • Incubation buffer (pH 7.5)
Final concentrationFor 250 mL
1% Triton X-100 2.5 mL of 100%
50 mM Tris-HCl12.5 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
H2O233 mL + 2 mL 2% NaN3
  • Staining solution, 100 mL: 40 mL of methanol, 10 mL of acetic acid, 50 mL of H2O, and 0.5 g of Coomassie Blue)
  • Destaining solution, 1 L: 400 mL of methanol, 100 mL of acetic acid, 500 mL of H2O.

Steps

1

Wash the gel 2 x 30 min with the washing buffer at room temperature with agitation.

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.

4

Stain the gel with staining solution for 30 min to 1 h at room temperature with agitation.

5

Rinse with water until excess staining solution is removed.

6

Incubate with destaining solution until bands can clearly be seen.

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