Product Pathways - Kinases
B-Raf Kinase #7351
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Description
Purified recombinant human B-Raf (Leu416-His766) kinase, supplied as a GST fusion protein.
Source / Purification
The GST-Kinase fusion protein was produced using a baculovirus expression system with a construct expressing human B-Raf (Leu416-His766) (GenBank Accession No. NM_004333) with an amino-terminal GST tag. The protein was purified by one-step affinity chromatography using glutathione-agarose.
Gel Staining
Figure 1. The purity of the GST-B-Raf fusion protein was analyzed using SDS/PAGE followed by Coomassie stain.
Kinase Assay - Radiometric
Figure 2. B-Raf kinase activity was measured in a radiometric assay using the following reaction conditions: 5 mM MOPS, pH 7.2, 2.5 mM β-glycerophosphate, 1 mM EGTA, 0.4 mM EDTA, 4 mM MgCl2, 0.05 mM DTT, 50 μM ATP, Substrate: Unactive MEK1 80 ng/μL, and variable amounts of B-Raf kinase.
Quality Control
The theoretical molecular weight of the GST-B-Raf fusion protein is 63 kDa. The purified kinase was quality controlled for purity using SDS-PAGE followed by Coomassie stain [Fig.1]. B-Raf kinase activity was determined using a radiometric assay [Fig.2].
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).
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Companion Products
This product is for in vitro research use only and is not intended for use in humans or animals. This product is not intended for use as therapeutic or in diagnostic procedures.
