Phospho-cRAF (Ser43) assay principle

The Phospho-cRAF (Ser43) assay measures cRAF when phosphorylated at Ser43. Unlike Western Blot, the assay is entirely plate-based and does not require gels, electrophoresis, or transfer. The assay uses 2 antibodies, one labeled with a donor fluorophore and 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 independently 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-cRAF (Ser43) two-plate assay protocol

The two-plate protocol involves culturing cells in a 96-well plate before lysis, then transferring lysates into a 384-well low volume detection plate before the addition of Phospho-cRAF (Ser43) HTRF detection reagents. This protocol allows for the cells' viability and confluence to be monitored.

Phospho-cRAF (Ser43) one-plate assay protocol

Detection of Phosphorylated cRAF (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 facilitates miniaturization while maintaining HTRF quality.

Specificity of phospho-cRAF (Ser43) assay using HAP-1 KO cell lines

The Phospho-cRAF (Ser43) protein levels were assessed with the HTRF Phospho-cRAF Ser43 kit in HAP1 cells (WT) and different HAP1 cell lines Knocked-Out for cRAF, ARAF and BRAF.
The different cell lines were plated in a 96-well plate (100,000 cells/well) and cultured for 24h. The cells were then stimulated with PMA (20µM, 15 min). After treatment, the cells were lysed with 50 µL of supplemented lysis buffer #2 for 30 minutes at RT under gentle shaking.

HTRF phospho-cRAF (Ser43) assay compared to Western Blot

HEK293 cells were grown in a T175 flask in complete culture medium at 37°C - 5% CO2 until 80% confluence. Cells were PMA stimulated (20µM, 15min) and lysed with 3 mL of supplemented lysis buffer#2 (1X) for 30 minutes at RT under gentle shaking.

cRAF signaling pathway

RAF proto-oncogene serine/threonine-protein kinase (c-RAF) is part of the MAP kinases pathway where it links the upstream effector Ras to the downstream MAPK/ERK cascade. It therefore plays 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.