MMP13 Recombinant Rabbit Monoclonal Antibody (3H13L17)

Protocol tips

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	-Rabbit (1-3 µg/mL)

Protocol tips
-Rabbit (1-3 µg/mL)

Publication protocol

Cells were seeded (7.5 × 104 cells/mL) on wells in a Corning flat-bottomed 24-well culture plate containing either natural or degraded ECM samples and were incubated for 5, 30, 60, 120, or 240 min at 37 °C and 5% CO2 in a serum-starving medium (DMEM supplemented with 2 mM l-glutamine, 1% penicillin/streptomycin, and 0.1% FBS). At the end of the incubation period, the ECM samples were removed carefully, washed with PBS, and then incubated in radioimmunoprecipitation assay (RIPA) buffer [20 mM Tris (pH 7.4), 137 mM NaCl, 10% glycerol, 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS, 2 mM EDTA, 1 mM PMSF] to produce cell lysates. Using this procedure, we could isolate the cell population that directly adhered to the natural or degraded ECM. Cell lysates then were analyzed by Western blotting using the indicated antibodies (60). Every experiment was performed three times in duplicate to test for reproducibility and to obtain statistically significant data. Quantifications of Western blot experiments were performed using ImageJ. Blots were scanned, and band densities were measured and quantified. The calculated ERK1/2 activity was measured as the ratio of band densities between pERK and ERK1/2 (pERK/ERK1/2).

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Manufacturer protocol

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