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2330
Raf Family Antibody Sampler Kit
Primary Antibodies

Raf Family Antibody Sampler Kit #2330

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Western blot analysis of extracts from Raji and HeLa cells, untreated or TPA-treated (30 minutes), using Phospho-A-Raf (Ser299) Antibody (upper) or A-Raf Antibody, #4432 (lower).

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

Western blot analysis of extracts from NIH3T3, HeLa and COS cells, untreated or treated with TPA, using Phospho-c-Raf (Ser338) (56A6) Rabbit mAb.

Western blot analysis of extracts from untreated or TPA-treated 293 and NIH/3T3 cells, using Phospho-c-Raf (Ser289/296/301) Antibody.

Western blot analysis of extracts from HeLa cells, untreated or TPA-treated, using Phospho-c-Raf (Ser259) Antibody (upper), or a total c-Raf antibody (lower).

Western blot analysis of extracts from various cell lines using c-Raf (D5X6R) Mouse mAb.

Western blot analysis of extracts from Raji and HeLa cells treated with TPA (200nM, 30 minutes) using Phospho-B-Raf (Ser445) Antibody (top) and total B-Raf Antibody (bottom).

Western blot analysis of extracts from HeLa, C2C12 and NBT-II cells, using B-Raf (55C6) Rabbit mAb.

After the primary antibody is bound to the target protein, a complex with HRP-linked secondary antibody is formed. The LumiGLO* is added and emits light during enzyme catalyzed decomposition.

Site specificity of Phospho-c-Raf (Ser259) Antibody: Western blot analysis of recombinant Myc-tagged c-Raf protein, wild-type (lanes 1 and 3) and S259A mutant (lanes 2 and 4), using Phospho-Raf (Ser259) Antibody or a Myc antibody. (Provided by Dr. Guri Tzivion, Massachusetts General Hospital.)

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Product Includes Quantity Applications Reactivity MW(kDa) Isotype
Phospho-A-Raf (Ser299) Antibody 4431 20 µl
  • WB
H M R 68 Rabbit 
A-Raf Antibody 4432 20 µl
  • WB
  • IP
H M R 68 Rabbit 
Phospho-c-Raf (Ser338) (56A6) Rabbit mAb 9427 20 µl
  • WB
H M R Mk 74 Rabbit IgG
Phospho-c-Raf (Ser289/296/301) Antibody 9431 20 µl
  • WB
H M 74 Rabbit 
Phospho-c-Raf (Ser259) Antibody 9421 20 µl
  • WB
  • IP
H M R Mk X 74 Rabbit 
c-Raf (D5X6R) Mouse mAb 12552 20 µl
  • WB
H M R Mk B Pg 75 Mouse IgG1
Phospho-B-Raf (Ser445) Antibody 2696 20 µl
  • WB
H M R Mk 86 Rabbit 
B-Raf (55C6) Rabbit mAb 9433 20 µl
  • WB
H M R Mk 86 Rabbit IgG
Anti-rabbit IgG, HRP-linked Antibody 7074 100 µl
  • WB
Goat 
Anti-mouse IgG, HRP-linked Antibody 7076 100 µl
  • WB
Horse 

Product Description

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 two 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 antibody is produced by immunizing rabbits with a synthetic peptide corresponding to residues close to the linker domain of human A-Raf. Polyclonal antibodies are purified by protein A and peptide affinity chromatography. The monoclonal antibodies are produced by immunizing animals with a synthetic phosphopeptide corresponding to residues surrounding Ser338 of human c-Raf, a synthetic peptide corresponding to human B-Raf and a recombinant protein corresponding to residues in the middle of human c-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 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).

  1. Avruch, J. et al. (1994) Trends Biochem Sci 19, 279-83.
  2. Chong, H. et al. (2001) EMBO J 20, 3716-27.
  3. King, A.J. et al. (1998) Nature 396, 180-3.
  4. Fabian, J.R. et al. (1993) Mol Cell Biol 13, 7170-9.
  5. Mason, C.S. et al. (1999) EMBO J 18, 2137-48.
  6. Zimmermann, S. and Moelling, K. (1999) Science 286, 1741-4.
  7. Sprenkle, A.B. et al. (1997) FEBS Lett 403, 254-8.
  8. Marais, R. et al. (1997) J Biol Chem 272, 4378-83.
  9. Guan, K.L. et al. (2000) J Biol Chem 275, 27354-9.
  10. Davies, H. et al. (2002) Nature 417, 949-54.
  11. Dougherty MK et al. (2005) Mol Cell 17, 215–24

Pathways & Proteins

Explore pathways + proteins related to this product.

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

Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.
U.S. Patent No. 7,429,487, foreign equivalents, and child patents deriving therefrom.