Product Pathways - MAPK Signaling
Raf Family Antibody Sampler Kit #2330
| Kit Includes | Quantity | Applications | Reactivity | 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 IP | 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 (L12G7) Mouse mAb #9434 | 40 µl | W IP F | H M R Mk Dm | 86 | Mouse IgG1 |
| Anti-rabbit IgG, HRP-linked Antibody #7074 | 100 µl | Goat | |||
| Anti-mouse IgG, HRP-linked Antibody #7076 | 100 µl | Horse |
Applications Key:
W=Western Blotting
IP=Immunoprecipitation
F=Flow Cytometry
Reactivity Key:
H=Human
M=Mouse
R=Rat
Mk=Monkey
C=Chicken
Dm=D. melanogaster
X=Xenopus
Dg=Dog
Species enclosed in parentheses are predicted to react based on 100% sequence homology.
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.
Western Blotting
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 Blotting
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 Blotting
Western blot analysis of extracts from HT29, NIH/3T3 and C6 cell lysates using A-Raf Antibody #4432.
Western Blotting
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 Blotting
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 Blotting
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 Blotting
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 Blotting
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 Blotting
Western blot analysis of extracts from K-562, HeLa, C1C12, NBT-II and COS cells using B-Raf (L12G7) Mouse mAb #9434.
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 four Western blot experiments.
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 antibody is produced by immunizing animals with a synthetic phosphopeptide corresponding to residues surrounding Ser338 of human c-Raf. The mouse monoclonal antibody is produced by immunizing animals with a recombinant fragment of 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 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).
- Avruch, J. et al. (1994) Trends Biochem. Sci. 19, 279-283.
- Chong, H. et al. (2001) EMBO J. 20, 3716-3727.
- King, A.J. et al. (1998) Nature 396, 180-183.
- Fabian, J.R. et al. (1993) Mol. Cell Biol. 13, 7170-7179.
- Mason, C.S. et al. (1999) EMBO J. 18, 2137-2148.
- Zimmermann, S. and Moelling, K. (1999) Science 286, 1741-1744.
- Sprenkle, A.B. et al. (1997) FEBS Lett. 403, 254-258.
- Marais, R. et al. (1997) J. Biol. Chem. 272, 4378-4383.
- Guan, K.L. et al. (2000) J. Biol. Chem. 275, 27354-27359.
- Davies, H. et al. (2002) Nature 417, 949-954.
- Dougherty, M.K. et al. (2005) Mol. Cell 17, 215-224.
Application References
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Protocols
- 2696:
- Western Blotting
- 4431:
- Western Blotting
- 4432:
- Immunoprecipitation, Western Blotting
- 9421:
- Immunoprecipitation, Western Blotting
- 9422:
- Immunoprecipitation, Western Blotting
- 9427:
- Western Blotting
- 9431:
- Western Blotting
- 9434:
- Flow, Immunoprecipitation, Western Blotting
Companion Products
- 4431 Phospho-A-Raf (Ser299) Antibody
- 4432 A-Raf Antibody
- 2696 Phospho-B-Raf (Ser445) Antibody
- 9433 B-Raf (55C6) Rabbit mAb
- 9434 B-Raf (L12G7) Mouse 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
This product is intended for research purposes only. The product is not intended to be used for therapeutic or diagnostic purposes in humans or animals.