Youssef Farhat, Written 3/1/14, Last updated 5/6/14
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Introduction
Gelatin zymography is an extremely sensitive and useful technique for measuring the relative amounts of active and inactive gelatinase (MMP-2 or MMP-9) in samples. However, as explained in our video on how to analyze gelatin zymograms (link below), an important limitations of gelatin zymography is the variation that can be observed from gel-to-gel.
The variation between zymograms may be due to a number of factors, including differences in incubation times, volumes of the various buffers, variations in the concentration of those buffers, and the age of zymograms as well as batch-to-batch variations between them.
This protocol is used to prepare a commercially available MMP-2 standard such that 10 ng of active MMP-2 can be added to each lane of a zymogram by loading 20 µL of the standard solution.
As shown in the video tutorial above, the MMP activity of our samples can then be normalized to the MMP activity of the standard, reducing the effect of gel-to-gel variations on our measurements of MMP activity.
If you are not sure how to identify which MMPs are in each band of your developed zymogram, click here to see our post on this subject.
Materials
- MMP-2, Proenzyme, Human, Recombinant, CHO Cells (EMD Millipore, #PF037)
- Novex Tris-Glycine SDS Sample Buffer (2X) (Invitrogen, #LC2676)
- 25 mM APMA in 0.1 M NaOH (may be prepared from a product like Millipore #164610-700MG)
- Incubator or water bath set to 37°C (a cell culture incubator works)
- Deionized water
- Normal lab supplies such as micropipettors, pipette tips, and 1.5 mL Eppendorf tubes
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Procedure
1. Aliquot and Store MMP-2
- Using deionized water as necessary, adjust the concentration of the MMP-2 standard to 100 ng/µL (Dissolve 10 µg MMP-2 into a total of 100 µL if not provided at that concentration).
- Aliquot the MMP-2 into 5 µL aliquots (each aliquot contains 500 ng of MMP-2) and store them at -80°C if they will not be used immediately.
2. Activate MMP-2 with APMA
- Add 43 µL of deionized water to the vial of MMP-2 containing 500 ng in 5 µL.
- Add 2 µL of 25 mM APMA to the vial and mix by pipetting (use a micropipettor set to ~25 µL).
- The vial now contains 50 µL of MMP-2 at a concentration of 10 ng/µL and APMA at a concentration of 1 mM.
- Incubate the vial at 37°C for 1 h to activate the MMPs.
3. Dilute the MMP-2 Standard to Working Concentration (0.5 ng/mL in Sample Buffer)
- Add 450 µL to the vial containing 50 µL of activated MMPs
- Add 500 µL of 2X Sample Buffer to the vial and vortex the solution
- The vial now contains 1 mL of ready-to-use active MMP-2 at a concentration of 0.5 ng/mL. By adding 20 µL of this solution to a lane in the zymogram, 10 ng of active MMP-2 will be loaded and use to normalize your data demonstrated in the video above.
- Aliquot the MMP-2 and freeze at -80°C for future use.
- I would aliquot 90 µL per tube, which is enough for 4 zymograms.
Related Protocols
- Farhat, Y. “Gelatin Zymography Protocol.” <http://protocol-place.com/assays/gelatin-zymography/gelatin-zymography-protocol/>
Citing this Protocol
Farhat, Y. “Preparing an Active MMP-2 Standard for Gelatin Zymography.” Protocol Place. Feb 2014. Accessed on mm/dd/yyyy. <http://protocol-place.com/>
