#4432
Store at -20°C
A-Raf Antibody
W, IP
Endogenous
H, M, R
68 kDa
Rabbit**
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 un-
derstood 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 hyperphos-
phorylation of these six sites is dependent on downstream
MEK signaling and renders c-Raf unresponsive to subse-
quent activation events (11).
Specificity/Sensitivity: A-Raf Antibody detects
endogenous levels of total A-Raf. This antibody does not
cross-react with c-Raf or B-Raf.
Source/Purification: Polyclonal antibodies are produced
by immunizing animals with a synthetic peptide (KLH-cou-
pled) corresponding to residues close to the linker domain
of human A-Raf. Antibodies are purified by protein A and
peptide affinity chromatography.
Storage: Supplied in 10 mM sodium HEPES (pH 7.5), 150 mM
NaCl, 100 µg/ml BSA and 50% glycerol. Store at –20°C.
Do not aliquot the antibody.
*Species cross-reactivity is determined by western blot.
** Anti-rabbit secondary antibodies must be used to
detect this antibody.
Recommended Antibody Dilutions:
Western Blotting
1:1000
Immunoprecipitation 1:50
For application specific protocols please see the web
page for this product at www.cellsignal.com.
Please visit www.cellsignal.com for a complete listing
of recommended companion products.
Orders
n
877-616-CELL (2355)
orders@cellsignal.com
Support
n
877-678-TECH (8324)
info@cellsignal.com
Web
n
www.cellsignal.com
Applications
Species Cross-Reactivity*
Molecular Wt.
Source
© 2010 Cell Signaling T
echnology
, Inc.
Background References:
(1) Avruch, J. et al. (1994) Trends Biochem. Sci. 19,
279–283.
(2) Chong, H. et al. (2001) EMBO J. 20, 3716–3727.
(3) King, A.J. et al. (1998) Nature 396, 180–183.
(4) Fabian, J.R. et al. (1993) Mol. Cell Biol. 13,
7170–7179.
(5) Mason, C.S. et al. (1999) EMBO J. 18, 2137–2148.
(6) Zimmermann, S. and Moelling, K. (1999) Science 286,
1741–1744.
(7) Sprenkle, A.B. et al. (1997) FEBS Lett. 403, 254–258.
(8) Marais, R. et al. (1997) J. Biol. Chem. 272,
4378–4383.
(9) Guan, K.L. et al. (2000) J. Biol. Chem. 275,
27354–27359.
(10) Davies, H. et al. (2002) Nature 417, 949–954.
(11) Dougherty, M.K. et al. (2005) Mol. Cell 17, 215–224.
IMPORTANT: For estern blots, incubate membrane with diluted antibody in 5% w/v BSA, 1X TBS, 0.1% Tween-20
at 4°C with gentle shaking, overnight.
kDa
140
100
80
C6
60
50
40
A-Raf
HT2
9
NIH/3T
3
Western blot analysis of extracts from HT-29, NIH-3T3 and
C6 cell lysates, using A-Raf Antibody.
Entrez-Gene ID #369
Swiss-Prot Acc. #P10398
Species Cross-Reactivity Key:
H—human
M—mouse
R—rat
Hm—hamster
Mk—monkey
Mi—mink
C—chicken
Dm—D. melanogaster X—Xenopus
Z—zebrafish
B—bovine
Dg—dog Pg—pig Sc—S. cerevisiae Ce—C. elegans
Hr—Horse
All—all species expected
Species enclosed in parentheses are predicted to react based on 100% homology.
Applications Key:
W—Western
IP—Immunoprecipitation
IHC—Immunohistochemistry
ChIP—Chromatin Immunoprecipitation
IF—Immunofluorescence
F—Flow cytometry
E-P—ELISA-Peptide
rev. 01/22/16
For Research Use Only. Not For Use In Diagnostic Procedures.