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.)
|Phospho-A-Raf (Ser299) Antibody 4431||20 µl||
||H M R||68||Rabbit|
|A-Raf Antibody 4432||20 µl||
||H M R||68||Rabbit|
|Phospho-c-Raf (Ser338) (56A6) Rabbit mAb 9427||20 µl||
||H M R Mk||74||Rabbit IgG|
|Phospho-c-Raf (Ser289/296/301) Antibody 9431||20 µl||
|Phospho-c-Raf (Ser259) Antibody 9421||20 µl||
||H M R Mk X||74||Rabbit|
|c-Raf (D5X6R) Mouse mAb 12552||20 µl||
||H M R Mk B Pg||75||Mouse IgG1|
|Phospho-B-Raf (Ser445) Antibody 2696||20 µl||
||H M R Mk||86||Rabbit|
|B-Raf (55C6) Rabbit mAb 9433||20 µl||
||H M R Mk||86||Rabbit IgG|
|Anti-rabbit IgG, HRP-linked Antibody 7074||100 µl||
|Anti-mouse IgG, HRP-linked Antibody 7076||100 µl||
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.
Each antibody in the Raf Family Antibody Sampler Kit recognizes only its specific target and does not cross react with other Raf family members.
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.
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).
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