20 μl of cell-free BMDM supernatant was added to 20 μl of Zymogram Sample Buffer (Bio-Rad) and loaded onto 10% polyacrylamide gels embedded with gelatin (Bio-Rad). Electrophoresis buffer was prepared from a 10× stock solution (15 g Tris base, 72 g Glycine, 5 g Sodium dodecyl sulfate dissolved in 500 ml Milli-Q water). Zymography gels were electrophoresed at 100 Volts for 1 hour. After electrophoresis, gels were removed from the cassette and incubated in 1× Zymogram Renaturation Buffer (Bio-Rad) at room temperature on a rocker plate for 30 minutes, followed by overnight incubation at 37°C in Zymography Development Buffer (Bio-Rad). Gels were then stained with 2.5% Coomassie Blue R-250 (Bio-Rad) dissolved in a solution of 50% methanol, 10% glacial acetic acid, and 50% Milli-Q water for 30 minutes. Gels were destained using 50% methanol, 10% glacial acetic acid, and 50% Milli-Q water, then imaged. Density analysis of the bands was performed using ImageJ. The appropriate position of the MMP-9 zymography band was verified using recombinant mouse MMP-9 (AnaSpec) (data not shown).
Recombinant mouse mmp 9
Manufactured by AnaSpec
Recombinant mouse MMP-9 is a laboratory reagent produced by AnaSpec. It is a recombinant form of the mouse matrix metalloproteinase-9 (MMP-9) protein. MMP-9 is an enzyme involved in the breakdown and remodeling of extracellular matrix components.
Automatically generated - may contain errors
Lab products found in correlation
Gelatin, by Bio-Rad (1 mentions)
Zymogram renaturation buffer, by Bio-Rad (1 mentions)
Zymogram sample buffer, by Bio-Rad (1 mentions)
Zymography development buffer, by Bio-Rad (1 mentions)
Coomassie blue r 250, by Bio-Rad (1 mentions)
Recombinant mouse rat mmp 2, by R&D Systems (1 mentions)
Photosmart 7520, by Adobe (1 mentions)
2 protocols using recombinant mouse mmp 9
1
Gelatin Zymography for MMP-9 Detection
2
Zymography for Detecting Matrix Metalloproteinases
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Samples were prepared as described above for immunoblot analysis. 45 μg of retinal protein extracts were mixed with zymogram loading buffer (Novex Tris Glycine SDS Sample buffer, Novex Life Technologies, Carlsbad, CA) without boiling and applied to 10% NOVEX Pre-Cast SDS polyacrylamide gel (Novex Life Technologies) in the presence of 0.1% gelatin under non-reducing conditions for electrophoresis. Positive controls for MMP-9 and MMP-2 included 1.5 ng of recombinant mouse MMP-9 (AnaSpec, Fremont, CA) and 6.6 ng of recombinant mouse/rat MMP-2 (R&D Systems, Minneapoilis, MN), respectively. After electrophoresis, the gels were washed with deionized water, and then each was incubated in zymogram renaturing buffer (Novex Life Technologies) for 30 minutes at room temperature and then developed in zymogram developing buffer (Novex Life Technologies) for 16 hours at 37°C to allow proteolysis of the substrates in the gels. After staining with SimpleBlue™ Safestain (Novex Life Technologies) for 1 hour, gels were de-stained in deionized water for 1 hour and imaged. Images were scanned using HP Photosmart 7520 and processed using Photoshop CC (Adobe, San Jose, CA) software.
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