Revision 9

#14994Store at -20C

Cell Signaling Technology

Orders: 877-616-CELL (2355) [email protected]

Support: 877-678-TECH (8324)

Web: [email protected] cellsignal.com

3 Trask LaneDanversMassachusetts01923USA
For Research Use Only. Not for Use in Diagnostic Procedures.
Applications:

WB, W-S, IP, IHC-P, IF-IC, FC-FP, ChIP, C&R

REACTIVITY:

H M R Mk

SENSITIVITY:

Endogenous

MW (kDa):

84, 91

Source/Isotype:

Rabbit IgG

UniProt ID:

#P42224

Entrez-Gene Id:

6772

Product Information

Product Usage Information

For optimal ChIP results, use 10 μl of antibody and 10 μg of chromatin (approximately 4 x 106 cells) per IP. This antibody has been validated using SimpleChIP® Enzymatic Chromatin IP Kits.

The CUT&RUN dilution was determined using CUT&RUN Assay Kit #86652.
Application Dilution
Western Blotting 1:1000
Simple Western™ 1:10 - 1:50
Immunoprecipitation 1:100
Immunohistochemistry (Paraffin) 1:1600 - 1:6400
Immunofluorescence (Immunocytochemistry) 1:200 - 1:800
Flow Cytometry (Fixed/Permeabilized) 1:100 - 1:400
Chromatin IP 1:50
CUT&RUN 1:50

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.

For a carrier free (BSA and azide free) version of this product see product #65748.

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.

Species Reactivity:

Human, Mouse, Rat, Monkey

Source / Purification

Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Pro688 of human Stat1 protein.

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.

  1. Heim, M.H. (1999) J Recept Signal Transduct Res 19, 75-120.
  2. Durbin, J.E. et al. (1996) Cell 84, 443-50.
  3. Meraz, M.A. et al. (1996) Cell 84, 431-42.
  4. Ihle, J.N. et al. (1994) Trends Biochem Sci 19, 222-7.
  5. Frank, D.A. (1999) Mol Med 5, 432-56.
  6. Wen, Z. et al. (1995) Cell 82, 241-50.

Species Reactivity

Species reactivity is determined by testing in at least one approved application (e.g., western blot).

Western Blot Buffer

IMPORTANT: For western blots, incubate membrane with diluted primary antibody in 5% w/v BSA, 1X TBS, 0.1% Tween® 20 at 4°C with gentle shaking, overnight.

Applications Key

WB: Western Blotting W-S: Simple Western™ IP: Immunoprecipitation IHC-P: Immunohistochemistry (Paraffin) IF-IC: Immunofluorescence (Immunocytochemistry) FC-FP: Flow Cytometry (Fixed/Permeabilized) ChIP: Chromatin IP C&R: CUT&RUN

Cross-Reactivity Key

H: human M: mouse R: rat Hm: hamster Mk: monkey Vir: virus 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 GP: Guinea Pig Rab: rabbit All: all species expected

Trademarks and Patents

Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.
Alexa Fluor is a registered trademark of Life Technologies Corporation.
All other trademarks are the property of their respective owners. Visit cellsignal.com/trademarks for more information.

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Revision 9
#14994

Stat1 (D1K9Y) Rabbit mAb

Western Blotting Image 1: Stat1 (D1K9Y) Rabbit mAb Expand Image
Western blot analysis of extracts from A549 cells (lane 1) or STAT1 knock-out cells (lane 2) using Stat1 (D1K9Y) Rabbit mAb #14994 (upper), and β-actin (D6A8) Rabbit mAb #8457 (lower). The absence of signal in the STAT1 knock-out A549 cells confirms specificity of the antibody for STAT1.
Western Blotting Image 2: Stat1 (D1K9Y) Rabbit mAb Expand Image
Western blot analysis of extracts from various cell lines using Stat1 (D1K9Y) Rabbit mAb.
Western Blotting Image 1: Stat1 (D1K9Y) Rabbit mAb Expand Image
Simple Western™ analysis of lysates (0.1 mg/mL) from MCF-7 cells using Stat1 (D1K9Y) Rabbit mAb #14994. The virtual lane view (left) shows the target band (as indicated) at 1:10 and 1:50 dilutions of primary antibody. The corresponding electropherogram view (right) plots chemiluminescence by molecular weight along the capillary at 1:10 (blue line) and 1:50 (green line) dilutions of primary antibody. This experiment was performed under reducing conditions on the Jess™ ​​​​​​​ Simple Western instrument from ProteinSimple, a BioTechne brand, using the 12-230 kDa separation module.
Immunoprecipitation Image 1: Stat1 (D1K9Y) Rabbit mAb Expand Image
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.
Immunohistochemistry Image 1: Stat1 (D1K9Y) Rabbit mAb Expand Image
Immunohistochemical analysis of paraffin-embedded human breast carcinoma using Stat1 (D1K9Y) Rabbit mAb.
Immunohistochemistry Image 2: Stat1 (D1K9Y) Rabbit mAb Expand Image
Immunohistochemical analysis of paraffin-embedded human lymphoma using Stat1 (D1K9Y) Rabbit mAb.
Immunofluorescence Image 1: Stat1 (D1K9Y) Rabbit mAb Expand Image
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).
Flow Cytometry Image 1: Stat1 (D1K9Y) Rabbit mAb Expand Image
Flow cytometric analysis of ACHN cells using Stat1 (D1K9Y) Rabbit mAb (solid line) compared to concentration-matched Rabbit (DA1E) mAb IgG XP® Isotype Control #3900 (dashed line). Anti-rabbit IgG (H+L), F(ab')2 Fragment (Alexa Fluor® 488 Conjugate) #4412 was used as a secondary antibody.
Chromatin Immunoprecipitation Image 1: Stat1 (D1K9Y) Rabbit mAb Expand Image
Chromatin immunoprecipitations were performed with cross-linked chromatin from HT-1080 cells treated with Human Interferon-γ (hIFN-γ) #8901 (50 ng/ml, 30 min) and either Stat1 (D1K9Y) Rabbit mAb or 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 α 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.
CUT and RUN Image 1: Stat1 (D1K9Y) Rabbit mAb Expand Image
CUT&RUN was performed with HT-1080 cells and Stat1 (D1K9Y) Rabbit mAb, using CUT&RUN Assay Kit #86652. DNA library was prepared using DNA Library Prep Kit for Illumina (ChIP-seq, CUT&RUN) #56795. The figure shows binding across FZD5 gene, a known target gene of Stat1 (see additional figure containing CUT&RUN-qPCR data).
CUT and RUN Image 2: Stat1 (D1K9Y) Rabbit mAb Expand Image
CUT&RUN was performed with HT-1080 cells treated with (hIFN-γ) #8901 (50 ng/ml, 30 min) and Stat1 (D1K9Y) Rabbit mAb or Rabbit (DA1E) mAb IgG XP® Isotype Control (CUT&RUN) #66362, using CUT&RUN Assay Kit #86652. The enriched DNA was quantified by real-time PCR using human AIM2 promoter primers, human FZD5 promoter primers, and SimpleChIP® Human α 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.
CUT and RUN Image 3: Stat1 (D1K9Y) Rabbit mAb Expand Image
CUT&RUN was performed with HT-1080 cells and Stat1 (D1K9Y) Rabbit mAb, using CUT&RUN Assay Kit #86652. DNA Libraries were prepared using DNA Library Prep Kit for Illumina (ChIP-seq, CUT&RUN) #56795. The figures show binding across chromosome 2 (upper), including FZD5 gene (lower), a known target gene of Stat1 (see additional figure containing CUT&RUN-qPCR data).