Revision 1
#2330
Store at -20C
Raf Family Antibody Sampler Kit
1 Kit
(8 x 20 microliters)
877-616-CELL (2355)
877-678-TECH (8324)
3 Trask Lane | Danvers | Massachusetts | 01923 | USA
For Research Use Only. Not for Use in Diagnostic Procedures.
Product Includes | Product # | Quantity | Mol. Wt | Isotype/Source |
---|---|---|---|---|
Phospho-A-Raf (Ser299) Antibody | 4431 | 20 µl | 68 kDa | Rabbit |
A-Raf Antibody | 4432 | 20 µl | 68 kDa | Rabbit |
Phospho-c-Raf (Ser338) (56A6) Rabbit mAb | 9427 | 20 µl | 74 kDa | Rabbit IgG |
Phospho-c-Raf (Ser289/296/301) Antibody | 9431 | 20 µl | 74 kDa | Rabbit |
Phospho-c-Raf (Ser259) Antibody | 9421 | 20 µl | 74 kDa | Rabbit |
c-Raf (D5X6R) Mouse mAb | 12552 | 20 µl | 75 kDa | Mouse IgG1 |
Phospho-B-Raf (Ser445) Antibody | 2696 | 20 µl | 86 kDa | Rabbit |
B-Raf (55C6) Rabbit mAb | 9433 | 20 µl | 86 kDa | Rabbit IgG |
Anti-rabbit IgG, HRP-linked Antibody | 7074 | 100 µl | Goat | |
Anti-mouse IgG, HRP-linked Antibody | 7076 | 100 µl | Horse |
Please visit cellsignal.com for individual component applications, species cross-reactivity, dilutions, protocols, and additional product information.
Description
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.
Storage
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 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).
Background References
- Avruch, J. et al. (1994) Trends Biochem Sci 19, 279-83.
- Chong, H. et al. (2001) EMBO J 20, 3716-27.
- King, A.J. et al. (1998) Nature 396, 180-3.
- Fabian, J.R. et al. (1993) Mol Cell Biol 13, 7170-9.
- Mason, C.S. et al. (1999) EMBO J 18, 2137-48.
- Zimmermann, S. and Moelling, K. (1999) Science 286, 1741-4.
- Sprenkle, A.B. et al. (1997) FEBS Lett 403, 254-8.
- Marais, R. et al. (1997) J Biol Chem 272, 4378-83.
- Guan, K.L. et al. (2000) J Biol Chem 275, 27354-9.
- Davies, H. et al. (2002) Nature 417, 949-54.
- Dougherty, M.K. et al. (2005) Mol Cell 17, 215-24.
Trademarks and Patents
Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.
All other trademarks are the property of their respective owners. Visit cellsignal.com/trademarks for more information.
Limited Uses
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Products are labeled with For Research Use Only or a similar labeling statement and have not been approved, cleared, or licensed by the FDA or other regulatory foreign or domestic entity, for any purpose. Customer shall not use any Product for any diagnostic or therapeutic purpose, or otherwise in any manner that conflicts with its labeling statement. Products sold or licensed by CST are provided for Customer as the end-user and solely for research and development uses. Any use of Product for diagnostic, prophylactic or therapeutic purposes, or any purchase of Product for resale (alone or as a component) or other commercial purpose, requires a separate license from CST. Customer shall (a) not sell, license, loan, donate or otherwise transfer or make available any Product to any third party, whether alone or in combination with other materials, or use the Products to manufacture any commercial products, (b) not copy, modify, reverse engineer, decompile, disassemble or otherwise attempt to discover the underlying structure or technology of the Products, or use the Products for the purpose of developing any products or services that would compete with CST products or services, (c) not alter or remove from the Products any trademarks, trade names, logos, patent or copyright notices or markings, (d) use the Products solely in accordance with CST Product Terms of Sale and any applicable documentation, and (e) comply with any license, terms of service or similar agreement with respect to any third party products or services used by Customer in connection with the Products.
Revision 1
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 Raji and HeLa cells, untreated or TPA-treated (30 minutes), using Phospho-A-Raf (Ser299) Antibody (upper) or A-Raf Antibody, #4432 (lower).
Revision 1
Western blot analysis of extracts from HT-29, NIH-3T3 and C6 cell lysates, using A-Raf Antibody.
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.
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.
Revision 1
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 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.
Revision 1
Western blot analysis of extracts from HeLa, C2C12 and NBT-II cells, using B-Raf (55C6) Rabbit mAb.
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.)