#14994 - Cell Signaling Technology

Store at
-20ºC
#14994
Stat1 (D1K9Y) Rabbit mAb
Support: +1-978-867-2388 (U.S.)
www.cellsignal.com/support
100 µl (10 western blots)
Orders: 877-616-2355 (U.S.)
[email protected]
Entrez-Gene ID #6772
UniProt ID #P42224
rev. 07/01/15
For Research Use Only. Not For Use In Diagnostic Procedures.
Applications
W, IP, IHC-P, ChIP, IF-IC, F
Endogenous
Species Cross-Reactivity*
Molecular Wt.
Isotype
H, M, R, Mk
84, 91 kDa
Rabbit IgG**
Storage: Supplied in 10 mM sodium HEPES (pH 7.5), 150
mM NaCl, 100 µg/ml BSA, 50% glycerol and less than 0.02%
sodium azide. Store at –20°C. Do not aliquot the antibody.
Specificity/Sensitivity: Stat1 (D1K9Y) Rabbit mAb recognizes endogenous levels of total Stat1 protein. This antibody
also cross-reacts with an unidentified protein of 150 kDa.
Source/Purification: Monoclonal antibody is produced by
immunizing animals with a synthetic peptide corresponding to
residues surrounding Pro688 of human Stat1 protein.
Background References:
(1) H
eim, M.H. (1999) J. Recept. Signal. Transduct. Res. 19,
75-120.
7
HT
-2
9
A1
72
A2
0
H4IIE
**Anti-rabbit secondary antibodies must be used to
detect this antibody.
CF
M
kDa
He
Background: The Stat1 transcription factor is activated in
response to a large number of ligands (1) and is essential for
responsiveness to IFN-α and IFN-γ (2,3). Phosphorylation of
Stat1 at Tyr701 induces Stat1 dimerization, nuclear translocation, and DNA binding (4). Stat1 protein exists as a pair of
isoforms, Stat1α (91 kDa) and the splice variant Stat1β (84
kDa). In most cells, both isoforms are activated by IFN-α, but
only Stat1α is activated by IFN-γ. The inappropriate activation
of Stat1 occurs in many tumors (5). In addition to tyrosine
phosphorylation, Stat1 is also phosphorylated at Ser727
through a p38 mitogen-activated protein kinase (MAPK)-dependent pathway in response to IFN-α and other cellular stresses
(6). Serine phosphorylation may be required for the maximal
induction of Stat1-mediated gene activation.
La
*Species cross-reactivity is determined by western blot.
200
140
100
80
Stat1
60
50
40
Western blot analysis of extracts from various cell lines using Stat1
(D1K9Y) Rabbit mAb.
Serum-starved
Recommended Antibody Dilutions:
Western blotting
1:1000
Immunoprecipitation1:100
Immunohistochemistry (Paraffin)
1:3200†
Unmasking buffer: Citrate
Antibody diluent: SignalStain® Antibody Diluent #8112
Detection reagent: SignalStain® Boost (HRP, Rabbit) #8114
†Optimal IHC dilutions determined using SignalStain® Boost
IHC Detection Reagent.
Immunofluorescence (IF-IC)1:400
IF Protocol:
Methanol Permeabilization required
Chromatin IP
1:50
Flow Cytometry
1:100
For product specific protocols and a complete listing
of recommended companion products please see the
product web page at www.cellsignal.com
IFN-a-treated
(2) D
urbin, J.E. et al. (1996) Cell 84, 443-50.
(3) M
eraz, M.A. et al. (1996) Cell 84, 431-42.
(4) Ihle, J.N. et al. (1994) Trends Biochem. Sci. 19, 222-7.
(5) F rank, D.A. (1999) Mol. Med. 5, 432-56.
(6) W
en, Z. et al. (1995) Cell 82, 241-250.
Confocal immunofluorescent analysis of HeLa cells, serum-starved overnight (left) or treated with
Human Interferon-α1 (hIFN-α1) #8927 (1,000 units/ml, 30 min; right), using Stat1 (D1K9Y)
Rabbit mAb (green) and β-Actin (8H10D10) Mouse mAb #3700 (red).
Alexa Fluor is a registered trademark of Life Technologies Corporation.
Tween is a registered trademark of ICI Americas, Inc.
IMPORTANT: For western blots, incubate membrane with diluted antibody in 5% w/v BSA, 1X TBS, 0.1%
Tween®20 at 4°C with gentle shaking, overnight.
Thank you for your recent purchase. If you would like
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www.cellsignal.com
© 2015 Cell Signaling Technology, Inc.
XP, SignalStain, SimpleChip and Cell Signaling Technology are trademarks of Cell Signaling Technology, Inc.
Applications: W—Western IP—Immunoprecipitation IHC—Immunohistochemistry ChIP—Chromatin Immunoprecipitation IF—Immunofluorescence F—Flow cytometry E-P—ELISA-Peptide Species Cross-Reactivity: 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.
2
Stat1 (D1K9Y) Rabbit mAb #14994
Normal Rabbit IgG #2729
3
100
Stat1
80
60
50
40
30
0.12
0.10
0.08
0.06
0.04
0.02
0
20
Events
1
Signal relative to input
kDa
200
140
IRF-1
TAP1
α Satellite
Immunoprecipitation of Stat1 from MCF7 cell extracts using
Rabbit (DA1E) mAb IgG XP® Isotype Control #3900 (lane 2) or
Stat1 (D1K9Y) Rabbit mAb (lane 3). Lane 1 is 10% input. Western blot was performed using Stat1 (9H2) Mouse mAb #9176.
Chromatin immunoprecipitations were performed with crosslinked chromatin from 4 x 10 6 HT-1080 cells treated with
Human Interferon-g (hIFN-g) #8901 (50 ng/ml, 30 min) and
either 10 μl of Stat1 (D1K9Y) Rabbit mAb or 2 μl of Normal
Rabbit IgG #2729 using SimpleChIP® Enzymatic Chromatin IP
Kit (Magnetic Beads) #9003. The enriched DNA was quantified by real-time PCR using human IRF-1 promoter primers,
SimpleChIP® Human TAP1 Promoter Primers #5148, and
SimpleChIP® Human a Satellite Repeat Primers #4486. The
amount of immunoprecipitated DNA in each sample is represented as signal relative to the total amount of input chromatin,
which is equivalent to one.
Immunohistochemical analysis of paraffin-embedded human
lymphoma using Stat1 (D1K9Y) Rabbit mAb.
Immunohistochemical analysis of paraffin-embedded human
breast carcinoma using Stat1 (D1K9Y) Rabbit mAb.
10
–
+
–
Rabbit (DA1E) mAb IgG
XP® Isotype Control
–
–
+
Stat1 (D1K9Y)
Rabbit mAb
Orders: 877-616-CELL (2355) | [email protected] | Support: 877-678-TECH (8324) | www.cellsignal.com/support
© 2015 Cell Signaling Technology, Inc.
Stat1
Flow cytometric analysis of ACHN cells using Stat1 (D1K9Y)
Rabbit mAb (blue) compared to concentration-matched Rabbit
(DA1E) mAb IgG XP® Isotype Control #3900 (red). Anti-rabbit
IgG (H+L), F(ab')2 Fragment (Alexa Fluor® 488 Conjugate)
#4412 was used as secondary antibody.