Protein samples were separated by SDS–polyacrylamide gel electrophoresis and were then transferred to a polyvinylidene difluoride membrane (Millipore). After blocking, blots were probed with the appropriate antibodies overnight at 4°C. After PBS wash, protein bands were developed using an ECL Prime Chemiluminescence kit (GE Healthcare) on a Bio-Rad developing and imaging platform.
Coimmunoprecipitation and Western Blot Analysis
Protein samples were separated by SDS–polyacrylamide gel electrophoresis and were then transferred to a polyvinylidene difluoride membrane (Millipore). After blocking, blots were probed with the appropriate antibodies overnight at 4°C. After PBS wash, protein bands were developed using an ECL Prime Chemiluminescence kit (GE Healthcare) on a Bio-Rad developing and imaging platform.
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Corresponding Organization : Emory University
Other organizations : University of South China, First Affiliated Hospital of University of South China, The University of Texas MD Anderson Cancer Center
Variable analysis
- Transfection of cells
- Protein complex formation and interactions
- Lysis buffer composition (1% NP-40, 150 mM NaCl, 2 mM EDTA, 50 mM Tris pH 8.0)
- Protease and phosphatase inhibitor cocktails
- Protein A beads for antibody-protein complex capture
- SDS-PAGE separation of protein samples
- Transfer of proteins to PVDF membrane
- Blocking of membrane
- Antibody incubation conditions (overnight at 4°C)
- PBS wash step
- ECL Prime Chemiluminescence kit for protein band development
- Positive control: Not explicitly mentioned.
- Negative control: Not explicitly mentioned.
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