B-RAF-V600E Cellular Phosphorylation Assay

B-RAF is a cytosolic serine-/threonine kinase that is mutated at a high frequency in human cancer. The constitutively active mutant B-RAF V600E is found in approximately 70% of human melanomas and many other cancers. B-RAF is a component of the conserved RAS-RAF-MEK-ERK-MAP kinase pathway that regulates cellular responses to extracellular growth signals. B-RAF interacts with the small G-protein Ras resulting in translocation to the plasma membrane and activating phosphorylation at Thr598 and Ser601. Subsequently, B-RAF phosphorylates MAPK-kinase MEK1 at Ser218 and Ser222, thereby activating MAP-kinases (ERKs) and downstream transcription factors that drive proliferation and survival. Inhibitors of B-RAF kinase activity such as Sorafenib (Nexavar, Bay43-9006) are developed for therapeutic application.

BRAF (V600E)

B-Raf, B-raf-1, BRAF1, RAFB1

Rat1

Rat1 cells were transduced with B-RAF mutant B-RAF V600E which exhibits constitutive kinase activity. B-RAF V600E was fused to the N-terminus of a modified estrogen receptor (ER) which allows B-RAF activity only in the presence of estrogen-analog Hydroxy-Tamoxifen (OHT) [1]. OHT-induced activity results in MEK1 phosphorylation and subsequent downstream effects such as ERK-phosphorylation, proliferation, and soft agar growth. MEK1 phosphorylation in lysates is detected by phospho-MEK1-Ser218/222-specific Sandwich-ELISA.

[1] Pritchard, CA et al. (1995) Mol Cell Biol 15, 6430.

Substrate phosphorylation as a readout of intracellular kinase activity via ELISA

Freiburg, Germany

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