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).

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

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.