Description
Product Usage Information
Western Blotting: 1:1000
Immunoprecipitation: 1:50
Storage
Supplied in 10 mM sodium HEPES (pH 7.5), 150 mM NaCl, 100 µg/ml BSA and 50% glycerol. Store at -20°C. Do not aliquot the antibody.
Protocol
Available protocols: Western Blotting, Immunoprecipitation
Specificity / Sensitivity
A-Raf Antibody detects endogenous levels of total A-Raf. This antibody does not cross-react with c-Raf or B-Raf.
Species Reactivity: Human, Mouse, Rat
Source / Purification
Polyclonal antibodies are produced by immunizing animals with a synthetic peptide corresponding to residues close to the linker domain of human A-Raf. Antibodies are purified by protein A and peptide affinity chromatography.
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).
Alternate Names
A-RAF; A-Raf proto-oncogene serine/threonine-protein kinase; A-Raf proto-oncogene, serine/threonine kinase; ARAF; ARAF1; Oncogene ARAF1; PKS; PKS2; Proto-oncogene A-Raf; Proto-oncogene A-Raf-1; Proto-oncogene Pks; RAFA1; Ras-binding protein DA-Raf; Serine/threonine-protein kinase A-Raf; v-raf murine sarcoma 3611 viral oncogene homolog; v-raf murine sarcoma 3611 viral oncogene homolog 1; v-raf murine sarcoma 3611 viral oncogene-like protein
