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Western blotting analysis

BS Beini Sun
HC Hongce Chen
XW Xiaoping Wang
TC Tongsheng Chen
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Cells (1.0 × 106 cells/well) were seeded in 6 well plates. Washed once with cold 1 × PBS, then incubation with RIPA lysate containing protease inhibitor and phosphatase inhibitor and obtain total protein. The supernatant was collected by centrifugation at 14,000 rmp for 10 min at 4 °C. Protein assay by bicinchoninic acid (BCA). Proteins were separated by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel and transferred to a 0.22 μm PVDF membrane. After incubation with 5% skim milk powder for 2 h at room temperature, the membranes were incubated with antibodies overnight at 4 °C. After washing three times with TBST, the membranes were incubated with HRP rabbit or mouse antibodies for 1.5 h at room temperature. The bands were visualized most frequently using ECL chemical substrate.

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