CST, 61231T, Phospho-B-Raf (Ser365) (F6P2S) Rabbit Monoclonal Antibody

Monoclonal Antibody for studying B-Raf (Ser365) phosphate. Validated for Western Blotting,Simple Western™,Immunoprecipitation. Available in 2 sizes. Highly specific and rigorously validated in-house, Phospho-B-Raf (Ser365) (F6P2S) Rabbit Monoclonal Antibody (CST #61231) is ready to ship. Product Usage Information Western Blotting: 1:1000 Simple Western™: 1:10 - 1:50 Immunoprecipitation: 1:50 Storage Supplied in 10 mM sodium HEPES (pH 7.5), 150 mM NaCl, 100 µg/mL BSA, 50% glycerol, and less than 0.02% sodium azide. Store at -20°C. Do not aliquot the antibody. Protocol Available protocols: Western Blotting, Immunoprecipitation Specificity / Sensitivity Phospho-B-Raf (Ser365) (F6P2S) Rabbit Monoclonal Antibody recognizes endogenous levels of B-Raf protein only when phosphorylated at Ser365. This antibody does not cross-react with B-Raf phosphorylated at Ser363, Ser364, or Ser446, or c-Raf phosphorylated at Ser259. This antibody detects a 70 kDa protein of unknown identity in some cell lines. Species Reactivity: Human, Mouse, Rat Source / Purification Monoclonal antibody is produced by immunizing animals with a synthetic phosphopeptide corresponding to residues surrounding Ser365 of human B-Raf protein. Background A-Raf, B-Raf, and c-Raf (Raf-1) are the main effectors recruited by GTP-bound Ras to activate the MEK-MAP kinase pathway (1). Activation of c-Raf is the best understood and involves phosphorylation at multiple activating sites, including Ser338, Tyr341, Thr491, Ser494, Ser497, and Ser499 (2). p21-activated kinase (PAK) has been shown to phosphorylate c-Raf at Ser338, and the Src family phosphorylates Tyr341 to induce c-Raf activity (3,4). Ser338 of c-Raf corresponds to similar sites in A-Raf (Ser299) and B-Raf (Ser446), although this site is constitutively phosphorylated in B-Raf (5). Inhibitory 14-3-3 binding sites on c-Raf (Ser259 and Ser621) can be phosphorylated by Akt and AMPK, respectively (6,7). While A-Raf, B-Raf, and c-Raf are similar in sequence and function, differential regulation has been observed (8). Of particular interest, B-Raf contains three consensus Akt phosphorylation sites (Ser365, Ser429, and Thr440) and lacks a site equivalent to Tyr341 of c-Raf (8,9). Research studies have shown that the B-Raf mutation V600E results in elevated kinase activity and is commonly found in malignant melanoma (10). Six residues of c-Raf (Ser29, Ser43, Ser289, Ser296, Ser301, and Ser642) become hyperphosphorylated in a manner consistent with c-Raf inactivation. The hyperphosphorylation of these six sites is dependent on downstream MEK signaling and renders c-Raf unresponsive to subsequent activation events (11). Phosphorylation of B-Raf at Ser365 by protein kinase A has an inhibitory effect on the RAS-RAF-MEK-ERK signaling pathway, similar to the phosphorylation of c-Raf at Ser259 (12, 13). Forskolin-induced phosphorylation of B-Raf Ser365 creates a docking site for 14-3-3 regulatory proteins, which competitively blocks binding of NRas to B-Raf, inhibiting downstream MEK and ERK activation (12). The 14-3-3 docking site can be disrupted in vitro by mutating Ser365 to alanine (12,13). Introducing the Ser365Ala mutation to B-Raf V600E cell lines promoted B-Raf self-dimerization, resulting in decreased sensitivity to Raf inhibitor PLX4720, prolonged MEK/ERK activation, and sustained proliferation (13). Alternate Names 94 kDa B-raf protein; B-raf; B-Raf proto-oncogene serine/threonine-protein kinase (p94); B-Raf proto-oncogene, serine/threonine kinase; B-Raf serine/threonine-protein; B-RAF1; BRAF; BRAF1; FLJ95109; MGC126806; MGC138284; murine sarcoma viral (v-raf) oncogene homolog B1; NS7; p94; Proto-oncogene B-Raf; RAFB1; Serine/threonine-protein kinase B-raf; v-raf murine sarcoma viral oncogene homolog B; v-Raf murine sarcoma viral oncogene homolog B1 Specification REACTIVITY: H M R SENSITIVITY: Endogenous MW (kDa): 86-96 Source/Isotype: Rabbit IgG