A-Raf Antibody

4432:

Immunoprecipitation , Western Blotting

A-Raf Antibody detects endogenous levels of total A-Raf. This antibody does not cross-react with c-Raf or B-Raf.

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.

Western Blotting

Western blot analysis of extracts from HT-29, NIH-3T3 and C6 cell lysates, using A-Raf Antibody.

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 protein 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 (Ser445), 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 (Ser364, Ser428 and Thr439) and lacks a site equivalent to Tyr341 of c-Raf (8,9). 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).

1. Avruch, J. et al. (1994) Trends Biochem. Sci. 19, 279-283.
2. Chong, H. et al. (2001) EMBO J. 20, 3716-3727.
3. King, A.J. et al. (1998) Nature 396, 180-183.
4. Fabian, J.R. et al. (1993) Mol. Cell Biol. 13, 7170-7179.
5. Mason, C.S. et al. (1999) EMBO J. 18, 2137-2148.
6. Zimmermann, S. and Moelling, K. (1999) Science 286, 1741-1744.
7. Sprenkle, A.B. et al. (1997) FEBS Lett. 403, 254-258.
8. Marais, R. et al. (1997) J. Biol. Chem. 272, 4378-4383.
9. Guan, K.L. et al. (2000) J. Biol. Chem. 275, 27354-27359.
10. Davies, H. et al. (2002) Nature 417, 949-954.
11. Dougherty, M.K. et al. (2005) Mol. Cell 17, 215-224.

• Tap, W.D. et al. (2010) Neoplasia 12, 637-49. Applications: Western Blotting

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• 4431 Phospho-A-Raf (Ser299) Antibody
• 2330 Raf Family Antibody Sampler Kit
• 9427 Phospho-c-Raf (Ser338) (56A6) Rabbit mAb
• 9422 c-Raf Antibody
• 9421 Phospho-c-Raf (Ser259) Antibody
• 2696 Phospho-B-Raf (Ser445) Antibody
• 9433 B-Raf (55C6) Rabbit mAb
• 7071 Phototope^® -HRP Western Blot Detection System, Anti-rabbit IgG, HRP-linked Antibody
• 7074 Anti-rabbit IgG, HRP-linked Antibody
• 7720 Prestained Protein Marker, Broad Range (Premixed Format)
• 7727 Biotinylated Protein Ladder Detection Pack
• 7003 20X LumiGLO^® Reagent and 20X Peroxide

For Research Use Only. Not For Use In Diagnostic Procedures.