- Home
- Protocols
-
Request a Protocol
Ask a
question
Total protein was extracted from cultured cells and analyzed as previously described [22]. Briefly, the whole cell lysates were prepared in RIPA buffer containing 1× protease inhibitor cocktail (Roche, Indianapolis, IN), 1 mM phenylmethylsulfonyl fluoride, 10 mM β-glycerophosphate and 10 mM NaF. The concentration of total protein was quantified by using Pierce™ BCA Protein Assay Kit (#23250, Thermo, USA). A total of 30 µg of protein extracts were loaded to 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to PVDF membranes (Millipore, USA). After blocking with 5% dried skimmed milk for 1 h at room temperature, the membrane was incubated with the primary antibodies overnight at 4 °C and then with HRP conjugated secondary antibodies (Promega, USA) for 1 h at room temperature, Protein bands were detected using enhanced chemiluminescence (ECL) detection reagent (Beyotime, Shanghai, China) on a FluorChem M system (Protein Simple, USA).
Do you have any questions about this protocol?
Post your question to gather feedback from the community. We will also invite the authors of this article to respond.
Post a Question
