Cells were lysed in the Radioimmunoprecipitation assay (RIPA) buffer as previously described (33 ). After lysis, the supernatant was collected. Equal amounts of protein (20 μg-40 μg) were run on for 1.5 hours at 130V using 4–20% Criterion TGX Stain-Free Protein Gels (Bio-Rad, Hercules, CA) and electro-transferred to a PVDF membrane by Iblot2 transfer stacks (Invitrogen, Waltham, MA). Transferred proteins were probed with both a Fli-1 polyclonal antibody described previously (30 (link)) and an antibody to β-actin (Cell Signaling, Beverly, MA). The results were visualized using the Odyssey Imaging System (LI-COR, Lincoln, NE).
Protocol detail
Protein Extraction and Western Blot Analysis
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Corresponding Organization :
Other organizations : Xiangya Hospital Central South University, Central South University, Medical University of South Carolina
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Variable analysis
independent variables
- None explicitly mentioned
- Protein expression levels of Fli-1 and β-actin
- Protein amount loaded (20 μg-40 μg)
- Electrophoresis time (1.5 hours at 130V)
- Protein gel type (4–20% Criterion TGX Stain-Free Protein Gels)
- Transfer method (electro-transferred to a PVDF membrane by Iblot2 transfer stacks)
- Positive control: Fli-1 polyclonal antibody described previously (30 (link))
- Negative control: β-actin antibody (Cell Signaling, Beverly, MA)
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