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MCF7 whole cell lysate was prepared by homogenization in modified RIPA buffer (150 mM NaCl, 50 mM Tris-HCl, pH 7.4, 1% Triton X-100, 1 mM EDTA, 1 mM PMSF,5 µg/ml Aprotinin, 1 µg/ml Pepstatin-A, 2 µg/ml Leupeptin, 1 mM Na3VO4, 1 mM NaF). Debris was removed by centrifugation. Protein concentration was determined with Bio-Rad protein assay. The lysate was boiled for 5 min in 1 x SDS sample buffer (50 mM Tris-HCl pH 6.8, 12.5% glycerol, 1% sodium dodecyl sulfate, 0.01% bromophenol blue) containing 5% b-mercaptoethanol.
Our Abpromise guarantee covers the use of ab3871 in the following tested applications.
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
|WB||Use at an assay dependent concentration. MCF7 whole cell lysate is ready to load on SDS-PAGE for Western blotting. We recommend loading 20 µg per lane for mini gels.|