Revision 14
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, ChIP-seq

REACTIVITY:

H M

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 and ChIP-seq results, use 5 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.

Application Dilution
Western Blotting 1:1000
Simple Western™ 1:10 - 1:50
Immunoprecipitation 1:100
Immunohistochemistry (Paraffin) 1:400 - 1:1600
Immunofluorescence (Immunocytochemistry) 1:200 - 1:800
Flow Cytometry (Fixed/Permeabilized) 1:100 - 1:400
Chromatin IP 1:100
Chromatin IP-seq 1:100

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 #88845.

Specificity / Sensitivity

Phospho-Stat1 (Tyr701) (58D6) Rabbit mAb detects endogenous levels of Stat1 only when phosphorylated at tyrosine 701. The antibody detects phosphorylated tyrosine 701 of p91 Stat1 and also the p84 splice variant. It does not cross-react with the corresponding phospho-tyrosines of other Stat proteins.

Species Reactivity:

Human, Mouse

Source / Purification

Monoclonal antibody is produced by immunizing animals with a synthetic phosphopeptide corresponding to residues surrounding Tyr701 of human Stat1.

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 ChIP-seq: Chromatin IP-seq

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.
All other trademarks are the property of their respective owners. Visit cellsignal.com/trademarks for more information.

限制使用

除非 CST 的合法授书代表以书面形式书行明确同意,否书以下条款适用于 CST、其关书方或分书商提供的书品。 任何书充本条款或与本条款不同的客书条款和条件,除非书 CST 的合法授书代表以书面形式书独接受, 否书均被拒书,并且无效。

专品专有“专供研究使用”的专专或专似的专专声明, 且未专得美国食品和专品管理局或其他外国或国内专管机专专专任何用途的批准、准专或专可。客专不得将任何专品用于任何专断或治专目的, 或以任何不符合专专声明的方式使用专品。CST 专售或专可的专品提供专作专最专用专的客专,且专用于研专用途。将专品用于专断、专防或治专目的, 或专专售(专独或作专专成)或其他商专目的而专专专品,均需要 CST 的专独专可。客专:(a) 不得专独或与其他材料专合向任何第三方出售、专可、 出借、捐专或以其他方式专专或提供任何专品,或使用专品制造任何商专专品,(b) 不得复制、修改、逆向工程、反专专、 反专专专品或以其他方式专专专专专品的基专专专或技专,或使用专品开专任何与 CST 的专品或服专专争的专品或服专, (c) 不得更改或专除专品上的任何商专、商品名称、徽专、专利或版专声明或专专,(d) 只能根据 CST 的专品专售条款和任何适用文档使用专品, (e) 专遵守客专与专品一起使用的任何第三方专品或服专的任何专可、服专条款或专似专专

Revision 14
#9167

Phospho-Stat1 (Tyr701) (58D6) Rabbit mAb

Western Blotting Image 1: Phospho-Stat1 (Tyr701) (58D6) Rabbit mAb Expand Image
使用 Phospho-Stat1 (Tyr701) (58D6) Rabbit mAb #9167(上图)、Stat1 (D1K9Y) Rabbit mAb #14994(中图)和 β-Actin (D6A8) Rabbit mAb #8457(下图),对未经处理或已经过 IFNa(#36000,100 ng/mL,5 分钟)或 IFNg(#80385,100ng/mL,30 分钟)处理的 HeLa 细胞提取物进行蛋白质印迹法分析。
Western Blotting Image 2: Phospho-Stat1 (Tyr701) (58D6) Rabbit mAb Expand Image
使用 Phospho-Stat1 (Tyr701) (58D6) Rabbit mAb #9167(上图)和 β-Actin (D6A8) Rabbit mAb #8457(下图),对未经处理或已经过 EGF(100 ng/mL,30 分钟)、IFNa(100 ng/mL,5 分钟)或 PDGF(100 ng/mL,5 分钟)处理的各种细胞系提取物进行蛋白质印迹法分析。
Western Blotting Image 3: Phospho-Stat1 (Tyr701) (58D6) Rabbit mAb Expand Image
使用 Phospho-Stat1 (Tyr701) (58D6) Rabbit mAb(上图)或 Stat1 Antibody (#9172)(下图),对未经处理或经干扰素 -α (IFN-α) 处理的 Hela 细胞的提取物进行蛋白质印迹分析。
Western Blotting Image 1: Phospho-Stat1 (Tyr701) (58D6) Rabbit mAb Expand Image
使用 Phospho-Stat1 (Tyr701) (58D6) Rabbit mAb #9167 对经过 IFN-α(100 ng/mL,5 分钟)处理的血清饥饿 HeLa 细胞的裂解物 (0.1 mg/mL) 进行 Simple Western™ 分析。虚拟泳道式图像(左图)显示一抗稀释比例为 1:10 和 1:50 时的单一靶标条带(如图所示)。对应的电泳图(右图)为一抗稀释比例在 1:10(蓝线)和 1:50(绿线)时沿毛细血管内分子量的化学发光结果。在还原条件下,使用 12-230 kDa 分离模块在 ProteinSimple(BioTechne 品牌)的 Jess™ Simple Western 仪器上进行该实验。
Immunoprecipitation Image 1: Phospho-Stat1 (Tyr701) (58D6) Rabbit mAb Expand Image
对经 IFNa(#36000,100 ng/mL,5 分钟)处理的 HeLa 细胞提取物中的磷酸化 Stat1 (Tyr701) 进行免疫沉淀法分析。泳道 1 为10% input,泳道 2 经 Rabbit (DA1E) mAb IgG XP® Isotype Control #3900 沉淀,而泳道 3 为 Phospho-Stat1 (Tyr701) (58D6) Rabbit mAb #9167。
Immunohistochemistry Image 1: Phospho-Stat1 (Tyr701) (58D6) Rabbit mAb Expand Image
Immunohistochemical analysis of paraffin-embedded Non-Hodgkin lymphoma control (left) or λ phosphatase treated (right), using Phospho-Stat1 (tyr701) (58D6) Rabbit mAb.
Immunohistochemistry Image 2: Phospho-Stat1 (Tyr701) (58D6) Rabbit mAb Expand Image
Immunohistochemical analysis of paraffin-embedded Non-Hodgkin lymphoma using Phospho-Stat1 (Tyr701) (58D6) Rabbit mAb #9167 in the presence of control peptide (left) or Phospho-Stat1 (Tyr701) Blocking Peptide (right).
Immunohistochemistry Image 3: Phospho-Stat1 (Tyr701) (58D6) Rabbit mAb Expand Image
使用 Phospho-Stat1 (Tyr701) (58D6) Rabbit mAb 对石蜡包埋的胃(慢性胃炎)细胞进行免疫组织化学分析。
Immunohistochemistry Image 4: Phospho-Stat1 (Tyr701) (58D6) Rabbit mAb Expand Image
使用 Phospho-Stat1 (Tyr701) (58D6) Rabbit mAb 在 SignalSlide® HeLa -/+ IFNa IHC Controls #55861 上(未经处理(左图)或经 Human Interferon-α1 (hIFN-α1) #8927(右图)处理的石蜡包埋的 Hela 细胞沉淀物)进行免疫组织化学分析。
Immunofluorescence Image 1: Phospho-Stat1 (Tyr701) (58D6) Rabbit mAb Expand Image
使用 Phospho-Stat1 (Tyr701) (58D6) Rabbit mAb(绿色)对未经处理(左图)或经 IFNα #9906(右图)处理的 Hela 细胞进行共聚焦免疫荧光分析。蓝色伪彩 = DRAQ5® #4084(DNA 荧光染料)。
Flow Cytometry Image 1: Phospho-Stat1 (Tyr701) (58D6) Rabbit mAb Expand Image
Flow cytometric analysis of U266B1 cells, untreated (blue) or treated with hIFN-β (100 ng/ml, 5 mins; green) using Phospho-Stat1 (Tyr701) (58D6) Rabbit mAb (solid lines) or concentration-matched Rabbit (DA1E) mAb IgG XP® Isotype Control #3900 (dashed lines). Anti-Rabbit IgG (H+L),F(ab')2 Fragment (Alexa Fluor® 488 Conjugate) #4412。
Flow Cytometry Image 2: Phospho-Stat1 (Tyr701) (58D6) Rabbit mAb Expand Image
使用 Phospho-Stat1 (Tyr701) (58D6) Rabbit mAb(实线)或浓度匹配的 Rabbit (DA1E) mAb IgG XP® Isotype Control #3900(虚线),对未经处理(蓝色)或已经 IFN-α(100ng/ml,5 分钟;绿色)处理的 Jurkat 细胞进行流式细胞分析。Anti-Rabbit IgG (H+L),F(ab')2 Fragment (Alexa Fluor® 488 Conjugate) #4412。
Chromatin Immunoprecipitation Image 1: Phospho-Stat1 (Tyr701) (58D6) Rabbit mAb Expand Image
使用 SimpleChIP® Plus Enzymatic Chromatin IP Kit (Magnetic Beads) #9005,对已经 IFN-γ(50 ng/ml,30分钟)处理的 HT-1080 细胞中提取的交联染色质,在加入 Phospho-Stat1 (Tyr701) (58D6) Rabbit mAb 后,进行染色质免疫沉淀。使用 DNA Library Prep Kit for Illumina® (ChIP-seq, CUT&RUN) #56795 制备 DNA 库。结果图显示在 IRF-1 内结合,IRF-1 是磷酸 Stat1 的一种已知靶标基因(参见包含 ChIP-qPCR 数据的其他结果图)。
Chromatin Immunoprecipitation Image 2: Phospho-Stat1 (Tyr701) (58D6) Rabbit mAb Expand Image
使用 SimpleChIP® Plus Enzymatic Chromatin IP Kit (Magnetic Beads) #9005,对已经 IFN-γ(50 ng/ml,30分钟)处理的 HT-1080 细胞中提取的交联染色质,在加入 Phospho-Stat1 (Tyr701) (58D6) Rabbit mAb 后,进行染色质免疫沉淀。使用 DNA Library Prep Kit for Illumina® (ChIP-seq, CUT&RUN) #56795 制备 DNA 库。结果图显示在染色体5(上图)内的结合,包括 Phospho-Stat1 的已知靶标基因 IRF-1(下图)(参见包含 ChIP-qPCR 数据的其他结果图)。
Chromatin Immunoprecipitation Image 3: Phospho-Stat1 (Tyr701) (58D6) Rabbit mAb Expand Image
使用 SimpleChIP® Plus Enzymatic Chromatin IP Kit (Magnetic Beads) #9005,对经 IFN-γ(50 ng/ml,30 分钟)处理的 HT-1080 细胞中提取的交联染色质,在加入 Phospho-Stat1 (Tyr701) (58D6) Rabbit mAb 或 Normal Rabbit IgG #2729 后,进行染色质免疫沉淀。使用 human IRF-1 promoter primers、SimpleChIP® Human TAP1 Promoter Primers #5148 和 SimpleChIP® Human α Satellite Repeat Primers #4486,通过实时 PCR 对富集的 DNA 进行定量分析。将每份样品中免疫沉淀的 DNA 的量表现为相对于所输入染色质总量(等于 1)的信号。