Phospho-c-RAF (Ser43) Assay principle

The Phospho-c-RAF (Ser43) assay measures c-RAF when phosphorylated at Ser43. Unlike Western Blot, the assay is entirely plate-based and does not require gels, electrophoresis, or transfer. The Phospho-c-RAF (Ser43) assay uses 2 labeled antibodies: one with a donor fluorophore, the other with an acceptor. The first antibody was selected for its specific binding to the phosphorylated motif on the protein, and the second for its ability to recognize the protein independent of its phosphorylation state. Protein phosphorylation enables an immune-complex formation involving both labeled antibodies and which brings the donor fluorophore into close proximity to the acceptor, thereby generating a FRET signal. Its intensity is directly proportional to the concentration of phosphorylated protein present in the sample, and provides a means of assessing the protein’s phosphorylation state under a no-wash assay format.

Phospho-c-RAF (Ser43) 2-plate Assay Protocol

The 2 plate protocol involves culturing cells in a 96-well plate before lysis. Then lysates are transferred into a 384-well low volume detection plate before the addition of Phospho-c-RAF (Ser43) HTRF detection reagents. This protocol enables the cells' viability and confluence to be monitored.

Phospho-c-RAF (Ser43) 1-plate assay protocol

Detection of Phosphorylated c-RAF (Ser43) with HTRF reagents can be performed in a single plate used for culturing, stimulation, and lysis. No washing steps are required. This HTS designed protocol enables miniaturization while maintaining robust HTRF quality.

Total and phospho c-RAF (S43) detection in TPA-stimulated HeLa cells

HeLa cells were plated under 100 µl in a 96-well plates (100,000 cells/well) in complete culture medium and incubated overnight at 37°C, 5% CO2. The day after, medium was removed and 50 µl of serum-free culture medium was added to starve cells for 5h at 37°C, 5% CO2. Then cells were treated with 50 µl of increasing concentrations of TPA for 15 min at 37°C, 5% CO2.

After medium removal, cells were then lysed with 50 µL of supplemented lysis buffer #2 for 30 minutes at RT under gentle shaking, and 16 µL of lysate were transferred into a low volume white microplate before the addition of 4 µL of the premixed HTRF phospho-cRaf (Ser43) or Total-cRaf detection reagents. The HTRF signal was recorded after ON incubation.

Total and phospho c-RAF (S43) detection in TPA-stimulated HEK293T cells

HEK293T cells were plated under 100 µl in a 96-well plates (100,000 cells/well) in complete culture medium and incubated overnight at 37°C, 5% CO2. The day after, medium was removed and 50 µl of serum-free culture medium was added to starve cells for 5h at 37°C, 5% CO2. Then cells were treated with 50 µl of increasing concentrations of TPA for 15 min at 37°C, 5% CO2.

After medium removal, cells were then lysed with 50 µL of supplemented lysis buffer #2 for 30 minutes at RT under gentle shaking, and 16 µL of lysate were transferred into a low volume white microplate before the addition of 4 µL of the premixed HTRF phospho-cRaf (Ser43) or Total-cRaf detection reagents. The HTRF signal was recorded after ON incubation.

Function and regulation of RSK1

RAF proto-oncogene serine/threonine-protein kinase (c-RAF) is par of the MAP kinases pathway where it links the upstream effector Ras to the downstream MAPK/ERK cascade. It therefore pays roles in cell fate decisions including proliferation, differentiation, apoptosis, survival and oncogenic transformation.

c-RAF is activated by Ser338 phosphorylation as a direct or indirect effect of PKC, or by GTP-bound Ras directly. Inactivation is ensured by the phosphorylation at Ser43 via MAPK/ERK-dependent feedback.