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Cat. # | Size | Qty. | Price |
---|---|---|---|
68776C | 1 Kit (96 assays) |
|
When ordering five or more kits, please contact us for processing time and pricing.
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REACTIVITY | H M R Mk |
Product Includes | Volume | Solution Color | |||
---|---|---|---|---|---|
c-Raf Rabbit mAb Coated Microwells | 96 tests | ||||
c-Raf Mouse Detection mAb | 1 ea | Red (Lyophilized) | |||
HRP Diluent | 5.5 ml | Red | |||
TMB Substrate 7004 | 11 ml | ||||
STOP Solution 7002 | 11 ml | ||||
Sealing Tape | 2 ea | ||||
ELISA Wash Buffer (20X) 9801 | 25 ml | ||||
Cell Lysis Buffer (10X) 9803 | 15 ml |
Product Information
NOTE: This protocol is for PathScan® kits that use an HRP directly conjugated to the detection antibody (Rapid Protocol), rather than a 2-step method where the detection antibody and a secondary-HRP are added sequentially.
NOTE: Prepare solutions with deionized/purified water or equivalent.
For adherent cells
For suspension cells
NOTE: Equilibrate all materials and prepared reagents to room temperature prior to running the assay.
NOTE: Initial color of positive reaction is blue, which changes to yellow upon addition of STOP Solution.
created July 2020
Protocol Id: 2144
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 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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