Protein samples were prepared by boiling the pelleted cells in radioimmunoprecipitation assay buffer, as described (41 (link)). The membranes were probed with 1:5000 mouse monoclonal anti-HA (16B12; Covance), 1:5000 rabbit polyclonal anti-FRB (from David Shore, University of Geneva), 1:5000 rabbit polyclonal anti-RAP1 (from David Shore, University of Geneva), 1:10,000 rabbit polyclonal anti–glyceraldehyde-3-phosphate dehydrogenase (A9521; Sigma-Aldrich), and 1:5000 mouse monoclonal anti-GFP antibodies (11814460001, Roche). Horseradish peroxidase–conjugated goat anti-mouse (Bio-Rad) or anti-rabbit (A8275, Sigma-Aldrich) immunoglobulin G were used at 1:10,000 as secondary antibodies.
Protocol detail
Western Blot Protein Analysis
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Anti antibodies
Antibodies
Buffer
Glyceraldehyde 3 phosphate dehydrogenase
Goat
Horseradish peroxidase
Immunoglobulin g
Membranes
Mouse
Protein
Rabbit
Radioimmunoprecipitation assay
Corresponding Organization :
Other organizations : University of Massachusetts Chan Medical School, University of Geneva
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Variable analysis
independent variables
- Boiling of pelleted cells in radioimmunoprecipitation assay buffer
- Expression levels of proteins probed, including HA, FRB, RAP1, GAPDH, and GFP
- Preparation of protein samples as described (
41 (link)) - Concentrations of primary antibodies used for probing the membranes (1:5000, 1:10,000)
- Concentrations of secondary antibodies used (1:10,000)
- Positive control: not explicitly mentioned
- Negative control: not explicitly mentioned
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