Recombinant mouse rat mmp 2

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.

Previously, detailed protocols for gelatin zymography were published [38 (link)]. 50 μg of retinal protein extracts were mixed with SDS Sample buffer excluding β-mercaptoethanol and boiling and then 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. The 6.6 ng of recombinant mouse/rat MMP2 (R&D Systems, Minneapolis, MN) and recombinant mouse MMP9 (R&D Systems, Minneapolis, MN) were used as positive controls. Following electrophoresis, the zymogram gels were washed with deionized water, and then each was incubated in renaturing buffer for 30 minutes at room temperature and then incubated in developing buffer (Novex Life Technologies) for 30 minutes at room temperature and further for 16 hours at 37°C. After staining with SimpleBlue^™ Safestain (Novex Life Technologies) for overnight, gels were de-stained in deionized water for overnight and imaged using HP Photosmart 7520 and processed using Photoshop (Adobe, San Jose, CA) software.