Youssef Farhat, Written 7/8/12, Last updated 5/6/14
Many biological processes are mediated by matrix metalloproteinases (MMPs), and evaluating their activity in model tissues such as collagen gels can be very useful.
Prior to performing gelatin zymography on collagen gel samples, cell-seeded collagen gels need to be lysed in order to release the MMPs into solution, clear away any unwanted debris, and maintain the activity of the gels.
To accomplish this, a Lysis Buffer (adapted from ) must be used which does not contain components that will inactivate the MMPs (such as EDTA).
In order to ensure equal loading of lanes, a protein assay such as the well-known BCA protein assay must be employed to measure the amount of protein in each sample.
- Zymography Lysis Buffer. This lysis buffer contains:
- 25 mM Tris-HCl, pH 7.5 (J.T. Baker, #4103-04)
- 100 mM NaCl (VWR, #BDH0286)
- 1% (volume/volume) IGEPAL CA-630 (Sigma-Aldrich, #I-3021)
- NaOH pellets (J.T. Baker, #3722)
- Protease inhibitors: 10 μg/mL aprotinin (Sigma-Aldrich, #A1153-1MG), 2 μg/mL leupeptin (Sigma-Aldrich, #L9783-5MG), and 4 mM benzamidine (Sigma-Aldrich, #B6506-5G)
- 5-mL Luer-lok syringes (BD, #309646)
- 20g 1½ needles (BD, #305176)
- Typical lab supplies and equipment such as a microcentrifuge, 1.5 mL Eppendorf tubes, cDNA tubes, pipettors, serological pipettes, micropipettors, multi-channel pipettor, reagent reservoirs, and pipette tips
1. Lysis Buffer Preparation
1) Prepare a 10% stock solution of IGEPAL CA-630 by mixing 10 mL with 90 mL of deionized water.
2) Prepare a 2 M NaCl stock solution by adding 11.688 g of NaCl to 100 mL of deionized water.
3) Prepare a 1 M Tris-HCl stock solution by adding 15.76 g to 100 mL of water. Using a pH probe, adjust the pH of the solution to 7.5 using tablets of NaOH (5-10 tablets worked for me). Be sure to check the molecular weight of the Tris-HCl is the same as the one used here (157.6 g/mol).
4) Combine 1.25 mL of Tris-HCl stock with 2.5 mL of NaCl stock, 5 mL of IGEPAL stock, and 41.25 mL of DI water. Sterile filter. Store at 4ºC.
5) Prepare protease-inhibitor stock solutions and aliquot as follows:
- Reconstitute the 1 mg of aprotinin in 200 mL of deionized water (5 mg/mL). Aliquot 22 mL per vial and store at -20ºC.
- Reconstitute the 5 mg of leupeptin in 2.5 mL of deionized water (2 mg/mL stock). Aliquot 12 mL per vial and store remainder at -20ºC.
- Measure 313 mg of leupeptin and reconstitute in 1 mL of deionized water (2 M stock). Aliquot 22 mL per vial and store at -20ºC.
6) Right before homogenization of samples, add protease inhibitors to the amount of zymography lysis buffer that will be used immediately. For 5 mL of lysis buffer, add 10 μL aprotinin, 5 μL leupeptin, and 10 μL benzamidine.
2. Sample Homogenization
1) Add 300 µL of cold lysis buffer to the collagen gel (the collagen gel samples I used in developing this protocol were 100-200 µL in volume and contained about 300,000 tendon fibroblast cells).
2) Vortex for 10 seconds.
3) Homogenize the samples on ice using Luer lock syringes and 20g needles.
4) Vortex for 5 seconds.
5) Let the samples rest on ice for 30 min.
6) Centrifuge the samples at 16,000 g for 10 min. Collect the supernatant and place into new Eppendorf tubes (be careful not to disturb any of the cell debris with the pipette tip).
1. Toth, M. and R. Fridman, Assessment of Gelatinases (MMP-2 and MMP-9 by Gelatin Zymography. Methods in molecular medicine, 2001. 57: p. 163-74.
Citing this Protocol