Kinexus Products

Target Full Name: B-Raf proto-oncogene serine-threonine-protein kinase; RafB proto-oncogene-encoded protein-serine kinase

Target Alias: B-RAF; B-RAF proto-oncogene serine/threonine-protein kinase; BRAF1; C-RMIL; Kinase B-Raf; P94; RAFB1; RMIL; RMIL serine/threonine-protein kinase; V-Raf murine sarcoma viral oncogene homolog B1; MGC126806; MGC138284; CCDS5863.1; ENSG00000157764

Product Type Specific: Raf-B protein kinase phosphosite-specific antibody

Antibody Code: PK534

Antibody Target Type: Phosphosite-specific

Antibody Phosphosite: S446+S447

Protein UniProt: P15056

Protein SigNET: Braf

Antibody Type: Polyclonal

Antibody Host Species: Rabbit

Antibody Immunogen Source: Human Raf-B (BRaf) sequence peptide Cat. No.: PE-04AHO99

Antibody Immunogen Sequence: RRD(pS)(pS)DDW(bA)C

Antibody Immunogen Description: Corresponds to amino acid residues R443 to W450; Just before the kinase catalytic domain. S446 and S447 phosphorylation stimulate phosphotransferase activity and induces interaction with H-Ras-1. These are two of the major sites of in vivo phosphorylation of B-Raf based on ≥130 and ≥154 reports, respectively, from high throughtput mass spectrometry studies recorded in PhosphoSitePlus. Raf-B is known to be phosphorylated in vitro at S446 by PAK1, and PKACa (PRKACA).

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: 0.5 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: Very strong immunoreactivity with immunogen peptide on dot blots.

Antibody Positive Control: The observed molecular mass of the processed target protein on SDS-PAGE gels is reported to be around 87-97 kDa.

Antibody Specificity: Very high

Antibody Cross Reactivity: No immunoreactivity on protein dot blots with recombinant human Raf1. No significant cross-reactive proteins detected in HEK-293, HeLa and TG98 cells, and in sea star oocytes.

Scientific Background: B-Raf (RAFB1) is a protein-serine/threonine kinase of the TKL group and RAF family. It is a cytoplasmic kinase that participates in the RAS>RAF>MEK>ERK pathways, which is involved in the transduction of mitogenic signals from the cell membrane to the nucleus and promotion of cell division, differentiation, and cytokine secretion. It features three conserved domains: CR1 (Ras-GTP-binding), CR2 (hinge region), and CR3 (catalytic kinase domain). Ras isoforms are inactive when bound to GDP, but when they are bound to GTP they become active and promote stimulation of B-Raf. Phosphorylation of Ser-446 increases phosphotransferase activity and induces interaction with H-Ras-1. Phosphorylation of Ser-579, Thr-599, and Ser-602 increases B-Raf phosphotransferase activity. Phosphorylation of Ser-729 increases phosphotransferase activity and induces interaction with 14-3-3 beta. Phosphorylation of Ser-365, Ser-429, and Thr-440 inhibits phosphotransferase activity. Phosphorylation of Thr-753 inhibits interaction with Raf1. B-Raf is expressed in most tissues with high expression in neuronal tissue. The transcription of several genes have been found to be increased by this pathway, including cyclins D1, D2 and D3, VEGF, c-myc, b3-integrin and mdm2. B-RAF is a known oncoprotein (OP). Cancer-related mutations in human tumours point to a gain of function of the protein kinase. Gain-of-function mutations in B-Raf are estimated to be present in ~15-19% of all human cancers, and 66% of malignant melanomas have been identified with a B-Raf somatic missense mutation. A single point mutation (V600E), located in the kinase catalytic domain in the activation loop between Subdomains VII and VIII. It has been reported to account for ~80% of the B-Raf mutations in human cancers. Mutated B-Raf proteins display constitutive elevated levels of phosphotransferase activity, resulting in the aberrant promotion of cell division, differentiation, and cytokine secretion. Beyond cancer, mutations in the BRAF gene are associated with developmental disorders such as Costello syndrome, Noonan syndrome, and cardiofaciocutaneous (CFC) syndrome. This description may include information annotated by UniProt and/or Google AI.

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