Product Name: Raf1-pS301+pT303

Product Number: AB-PK792

Size:	25 µg		Price:	89.00
			$US

Target Full Name: Raf1 proto-oncogene-encoded protein-serine kinase

Target Alias: cRaf; c-Raf; c-Raf; Kinase Raf1; Murine leukemia viral (V-raf-1) oncogene homologue 1; NS5; Protein kinase raf 1; Raf proto-oncogene serine/threonine-protein kinase; Raf1; V-raf-1 murine leukemia viral oncogene 1; CCDS2612.1; ENSG00000132155; B4E1N6; Q59HC8

Product Type Specific: Protein kinase phosphosite-specific antibody

Antibody Code: PK792

Antibody Target Type: Phosphosite-specific

Antibody Phosphosite: S301+T303

Protein UniProt: P04049

Protein SigNET: P04049

Antibody Type: Polyclonal

Antibody Host Species: Rabbit

Antibody Immunogen Source: Human Raf1 (RafC) sequence peptide Cat. No.: PE-04AHS90

Antibody Immunogen Sequence: NNL(pS)P(pT)GW(bA)C

Antibody Immunogen Description: Corresponds to amino acid residues N298 to W305; In the region between the C1_1 and kinase catalytic domains. This is the major in vivo phosphorylation sites in Raf1.

Production Method: The immunizing peptide was produced by solid phase synthesis on a multipep peptide synthesizer and purified by reverse-phase hplc chromatography. Purity was assessed by analytical hplc and the amino acid sequence confirmed by mass spectrometry analysis. This peptide was coupled to KLH prior to immunization into rabbits. New Zealand White rabbits were subcutaneously injected with KLH-coupled immunizing peptide every 4 weeks for 4 months. The sera from these animals was applied onto an agarose column to which the immunogen peptide was thio-linked. Antibody was eluted from the column with 0.1 M glycine, pH 2.5. Subsequently, the antibody solution was neutralized to pH 7.0 with saturated Tris.This antibody was also subject to negative purification over phosphotyrosine-agarose.

Antibody Modification: Unconjugated. Contact KInexus if you are interest in having the antibody biotinylated or coupled with fluorescent dyes.

Antibody Concentration: 0.75 mg/ml

Storage Buffer: Phosphate buffered saline pH 7.4, 0.05% Thimerasol

Storage Conditions: For long term storage, keep frozen at -40°C or lower. Stock solution can be kept at +4°C for more than 3 months. Avoid repeated freeze-thaw cycles.

Product Use: Western blotting | Antibody microarray

Antibody Dilution Recommended: 2 µg/ml for immunoblotting

Antibody Potency: Medium immunoreactivity of a target-sized protein by Western blotting in MCF7 cells, but was not insulin responsive. Also weak detection in HepG2 cells. Very strong immunoreactivity with immunogen peptide on dot blots.

Antibody Species Reactivity: Human

Antibody Positive Control: The observed molecular mass of the processed target protein on SDS-PAGE gels is reported to be around 70-75 kDa.

Antibody Specificity: Very high

Antibody Cross Reactivity: No significant cross-reactive proteins detected in phenylarsine oxide (PAO)+vanadate-treated HeLa cells, EGF-treated A431 cells and insulin-treated MCF7 cells, when these cells were homogenized in SDS-PAGE sample buffer.

Related Product 1: Raf1-pS301+pT303 blocking peptide

Related Product 2: Raf1-III (Raf1-4) pan-specific antibody (Cat. No.: AB-NK155-2)

Related Product 3: Raf1-C2 (Raf1-5) pan-specific antibody (Cat. No.: AB-NK155-3)

Scientific Background: Raf1 is a protein-serine/threonine kinase of the TKL group and Raf family. It links signalling from activated membrane-associated Ras GTPases with the MAPK/ERK intracellular signalling cascade to control the differentiation, proliferation, survival, apoptosis, and oncogenic transformation of cells. Raf1 has anti-apoptotic functions through its phosphorylation of S75 of the BAD/Bcl2-antagonist of cell death. Bcl2 then targets Raf1 to the mitochondria and once activated, Raf1 enhances Bcl2 mediated apoptotic resistance. Raf1 is activated by direct binding to Ras isforms and phosphorylation at S338, S339, Y340, Y341, S471, T481, T491, S494, S497, S499 and S621. Phosphorylation of S471 induces interaction with MEK1, and. phosphorylation at S621 induces interaction with 14-3-3-zeta. Phosphorylation at S233 and S289 inhibits interaction with H-Ras-1 and increases interaction with 14-3-3 beta. Phosphorylation at S259 inhibits phosphotransferase activity, reduces H-Ras-1 and PPP2CB binding, and induces binding of 14-3-3-beta and zeta. It also appears to necessary for PKC-alpha activation of Raf1. Phosphorylation of S43, S296, S301 and S642 inhibits phosphotransferase activity and inhibits interaction with H-Ras-1. Phosphorylation of Y341 inhibits interaction with Raf1. Raf1 appears to be an oncoprotein (OP), and cancer-related mutations in human tumours point to a gain of function of the protein kinase. Overactive Raf-1 kinase phosphotransferase activity due to different mutations has been implicated in the pathogenesis of both Noonan and Leopard syndromes. Substitution mutations are also associated with other cancers. Inhibition of Raf1 phosphotransferase activity by the protein phosphatase PPP5C is impaired (no binding) with S338D and S339E mutations when these occur in conjunction with phosphomimetic Y340D and Y341D mutations in Raf1. This results in a constitutively active kinase, and extensive phosphorylation of S338. The T491D and T494D can increase kinase phosphotransferase activity but the kinase is still sensitive to PPP5C. Methylation of Raf1 can be inhibited with a R563K mutation, leading to increased stability and catalytic activity. Raf1 is also upregulated in many cancers, since Ras (an upstream activator of Raf) is mutated/upregulated in many cancers. For example, Raf1 is over-expressed due to oncogenic ras mutations in about 35% of Non-Small Cell Lung Cancer (NSCLC). A small number of Raf mutations have been found in stomach and mouth tumours.

Figure 1. Epitope mapping of Raf1-pS301+pT303 antibody with similar phosphopeptides on dot blots.

Figure 2. Identification of phosphosites related to Raf1-pS301+pT303.