Product Pathways - MAPK Signaling
Raf Family Antibody Sampler Kit #2330
|2330S||1 Kit (8 x 40 µl)||---||In Stock||---|
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|Kit Includes||Quantity||Applications||Reactivity||Homology†||MW (kDa)||Isotype|
|Phospho-A-Raf (Ser299) Antibody #4431||40 µl||W||H, M, R||68||Rabbit|
|A-Raf Antibody #4432||40 µl||W, IP||H, M, R||68||Rabbit|
|Phospho-c-Raf (Ser338) (56A6) Rabbit mAb #9427||40 µl||W||H, M, R, Mk||74||Rabbit IgG|
|Phospho-c-Raf (Ser289/296/301) Antibody #9431||40 µl||W||H, M||R||74||Rabbit|
|Phospho-c-Raf (Ser259) Antibody #9421||40 µl||W, IP||H, M, R, X||C||74||Rabbit|
|c-Raf Antibody #9422||40 µl||W||H, M, R, Mk||65 to 75||Rabbit|
|Phospho-B-Raf (Ser445) Antibody #2696||40 µl||W||H, M, R, Mk||C, Dg||86||Rabbit|
|B-Raf (55C6) Rabbit mAb #9433||40 µl||W||H, M, R, Mk||86||Rabbit IgG|
|Anti-rabbit IgG, HRP-linked Antibody #7074||100 µl||Goat|
†Species predicted to react based on 100% sequence homology.
Applications Key: W=Western Blotting, IP=Immunoprecipitation
Reactivity Key: H=Human, M=Mouse, R=Rat, Mk=Monkey, X=Xenopus
Western blot analysis of extracts from Raji and HeLa cells, untreated or TPA-treated (200 nM, 30 minutes) using Phospho-B-Raf (Ser445) Antibody #2696 (top) and total B-Raf Antibody (bottom).
Western blot analysis of extracts from Raji and HeLa cells, untreated or TPA-treated (30 minutes), using Phospho-A-Raf (Ser299) Antibody #4431 (upper) or A-Raf Antibody #4432 (lower).
Western blot analysis of extracts from HT29, NIH/3T3 and C6 cell lysates using A-Raf Antibody #4432.
Western blot analysis of recombinant Myc-tagged c-Raf protein, wildtype (lanes 1 and 3) and S259A mutant (lanes 2 and 4), using Phospho-Raf (Ser259) Antibody #9421 or a Myc antibody (provided by Dr. Guri Tzivion, Massachusetts General Hospital).
Western blot analysis of extracts from HeLa cells, untreated or TPA-treated, using Phospho-c-Raf (Ser259) Antibody #9421 (upper), or a total c-Raf antibody (lower).
Western blot analysis of extracts from 293 cells, untreated or TPA-treated (200 nM for 30 minutes), using c-Raf Antibody #9422 (lower) or Phospho-c-Raf (Ser338) Rabbit mAb #9427 (upper).
Western blot analysis of extracts from NIH3T3, HeLa and COS cells, untreated or treated with TPA using Phospho-c-Raf (Ser338) (56A6) Rabbit mAb #9427.
Western blot analysis of extracts from untreated or TPA-treated 293 and NIH/3T3 cells using Phospho-c-Raf (Ser289/296/301) Antibody #9431.
Western blot analysis of extracts from HeLa, C2C12 and NBT-II cells, using B-Raf (55C6) Rabbit mAb #9433.
The Raf Family Antibody Sampler Kit provides a fast and economical means to investigate Raf signaling. The kit contains enough primary and secondary antibody to perform four Western blot experiments.
Specificity / Sensitivity
Each antibody in the Raf Family Antibody Sampler Kit recognizes only its specific target and does not cross react with other Raf family members.
Source / Purification
The phospho-specific polyclonal antibodies are produced by immunizing rabbits with a synthetic phosphopeptide corresponding to residues surrounding Ser299 of human A-Raf, Ser445 of human B-Raf and Ser259, 289, 296 and 301 of c-Raf. The total polyclonal antibodies are produced by immunizing rabbits with a synthetic peptide corresponding to residues close to the linker domain of human A-Raf and a region surrounding Pro302 of human c-Raf. Polyclonal antibodies are purified by protein A and peptide affinity chromatography. The rabbit monoclonal antibodies are produced by immunizing animals with a synthetic phosphopeptide corresponding to residues surrounding Ser338 of human c-Raf and a synthetic peptide corresponding to human B-Raf.
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). 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).
- Avruch, J. et al. (1994) Trends Biochem Sci 19, 279-83.
- Chong, H. et al. (2001) EMBO J 20, 3716-27.
- King, A.J. et al. (1998) Nature 396, 180-3.
- Fabian, J.R. et al. (1993) Mol Cell Biol 13, 7170-9.
- Mason, C.S. et al. (1999) EMBO J 18, 2137-48.
- Zimmermann, S. and Moelling, K. (1999) Science 286, 1741-4.
- Sprenkle, A.B. et al. (1997) FEBS Lett 403, 254-8.
- Marais, R. et al. (1997) J Biol Chem 272, 4378-83.
- Guan, K.L. et al. (2000) J Biol Chem 275, 27354-9.
- Davies, H. et al. (2002) Nature 417, 949-54.
- Dougherty, M.K. et al. (2005) Mol Cell 17, 215-24.
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- 4431 Phospho-A-Raf (Ser299) Antibody
- 4432 A-Raf Antibody
- 2696 Phospho-B-Raf (Ser445) Antibody
- 9433 B-Raf (55C6) Rabbit mAb
- 9421 Phospho-c-Raf (Ser259) Antibody
- 9431 Phospho-c-Raf (Ser289/296/301) Antibody
- 9427 Phospho-c-Raf (Ser338) (56A6) Rabbit mAb
- 9422 c-Raf Antibody
- 7074 Anti-rabbit IgG, HRP-linked Antibody
- 7076 Anti-mouse IgG, HRP-linked Antibody
- 7075 Anti-biotin, HRP-linked Antibody
- 7727 Biotinylated Protein Ladder Detection Pack
- 7003 20X LumiGLO® Reagent and 20X Peroxide
- 9160 MEK1/2 HeLa Control Cell Extracts
For Research Use Only. Not For Use In Diagnostic Procedures.
Cell Signaling Technology® is a trademark of Cell Signaling Technology, Inc.
Select rabbit monoclonal antibodies are developed, validated, and produced at CST using in part technology under license (granting certain rights including those under U.S. Patents No. 5,675,063 and in some instances 7,429,487) from Epitomics, Inc.