Youssef Farhat, Written 8/15/12, Last updated 5/6/14
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Introduction
This protocol describes how to prepare monolayer cell lysates for MMP analysis with gelatin [1], casein or collagen [2] zymography. Home-made lysis buffer is described and protease inhibitors are added just before lysis of the samples.
Materials
- Zymography Lysis Buffer (Recipe adapted from reference [1]). 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)
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- Typical lab supplies and equipment such as a microcentrifuge, 1.5 mL Eppendorf tubes, cDNA tubes, pipettors, serological pipettes, micropipettors, and pipette tips
Procedure
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) Remove the culture media from the cells. If performing zymography on the media samples as well, collect some into 1.5 mL Eppendorf tubes and centrifuge at 1,000 g for 10 min to pellet any cells or debris and then collect the supernatant.
2) Wash the cells twice with cold PBS (use the same volume as the culture media that was removed).
3) Add 300 μL of cold lysis buffer to the 100 mm dishes.
4) Using a rubber policeman, scrape the cells from the plate surface into the lysis buffer and incubate on ice for at least 15 min.
5) Collect the lysates into 1.5 mL Eppendorf tubes.
6) Vortex the samples for 5 seconds.
7) Centrifuge the samples at 16,000 g for 20 min at 4°C.
8) Collect the supernatant and measure protein concentration or store samples at -80°C if they will not be assayed immediately.
References
1. Toth M, Fridman R (2001) Assessment of Gelatinases (MMP-2 and MMP-9 by Gelatin Zymography. Methods in molecular medicine 57: 163-174.
2. Gogly B, Groult N, Hornebeck W, Godeau G, Pellat B (1998) Collagen zymography as a sensitive and specific technique for the determination of subpicogram levels of interstitial collagenase. Analytical Biochemistry 255: 211-216.
Citing this Protocol
Farhat, Y. “Lysis of Monolayer Samples for Zymography.” The Protocol Place. Aug 2012. Accessed on mm/dd/yyyy. <http://protocol-place.com/>
