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.

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 9
#14994

Stat1 (D1K9Y) Rabbit mAb

Western Blotting Image 1: Stat1 (D1K9Y) Rabbit mAb Expand Image
使用 Stat1 (D1K9Y) Rabbit mAb #14994(上图)和 β-actin (D6A8) Rabbit mAb #8457(下图)对 A549 细胞(泳道 1)或 STAT1 敲除型细胞(泳道 2)的提取物进行蛋白印迹分析。STAT1 敲除型 A549 细胞中没有信号,这证实了抗体对 STAT1 的特异性。
Western Blotting Image 2: Stat1 (D1K9Y) Rabbit mAb Expand Image
使用 Stat1 (D1K9Y) Rabbit mAb 对不同细胞系的提取物进行蛋白质印迹分析。
Western Blotting Image 1: Stat1 (D1K9Y) Rabbit mAb Expand Image
使用 Stat1 (D1K9Y) Rabbit mAb #14994 对 MCF-7 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: Stat1 (D1K9Y) Rabbit mAb Expand Image
使用 Rabbit (DA1E) mAb IgG XP® Isotype Control #3900 (泳道 2) 或 Stat1 (D1K9Y) Rabbit mAb (泳道 3),对MCF7 细胞提取物的 Stat1 进行免疫沉淀。泳道 1 是 10% 输入对照。使用 Stat1 (9H2) Mouse mAb #9176 进行蛋白质印迹分析。
Immunohistochemistry Image 1: Stat1 (D1K9Y) Rabbit mAb Expand Image
使用 Stat1 (D1K9Y) Rabbit mAb 对石蜡包埋的人乳腺癌组织进行免疫组织化学分析。
Immunohistochemistry Image 2: Stat1 (D1K9Y) Rabbit mAb Expand Image
使用 Stat1 (D1K9Y) Rabbit mAb 对石蜡包埋的人淋巴瘤组织进行免疫组织化学分析。
Immunofluorescence Image 1: Stat1 (D1K9Y) Rabbit mAb Expand Image
使用 Stat1 (D1K9Y) Rabbit mAb (绿色) 与 β-Actin (8H10D10) Mouse mAb #3700 (红色),对经过血清饥饿过夜(左图)处理,或经Human Interferon-α1 (hIFN-α1) #8927 (1,000 单位/mL,30 分钟(右图) 处理的 HeLa 细胞进行共聚焦免疫荧光分析。
Flow Cytometry Image 1: Stat1 (D1K9Y) Rabbit mAb Expand Image
以 Rabbit (DA1E) mAb IgG XP® Isotype Control #3900(虚线)作为对照,使用 Stat1 (D1K9Y) Rabbit mAb(实线)对 ACHN 细胞进行流式细胞分析。Anti-rabbit IgG (H+L)、F(ab')2 Fragment (Alexa Fluor® 488 Conjugate) #4412 作为二抗。
Chromatin Immunoprecipitation Image 1: Stat1 (D1K9Y) Rabbit mAb Expand Image
使用 SimpleChIP® Enzymatic Chromatin IP Kit (Magnetic Beads) #9003,对经 Human Interferon-γ (hIFN-γ) #8901(50 ng/ml,30 分钟)处理的 HT-1080 细胞的交联染色质,与 Stat1 (D1K9Y) Rabbit mAb 或 Normal Rabbit IgG #2729 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。
CUT and RUN Image 1: Stat1 (D1K9Y) Rabbit mAb Expand Image
使用 CUT&RUN Assay Kit #86652 对经过 (hIFN-γ) #8901(50 ng/ml,30 分钟)处理的 HT-1080 细胞和 Stat1 (D1K9Y) Rabbit mAb 或 Rabbit (DA1E) mAb IgG XP® Isotype Control (CUT&RUN) #66362 进行 CUT&RUN 分析。使用 human AIM2 promoter primers、human FZD5 promoter primers 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。
CUT and RUN Image 2: Stat1 (D1K9Y) Rabbit mAb Expand Image
使用 CUT&RUN Assay Kit #86652 对 HT-1080 细胞和 Stat1 (D1K9Y) Rabbit mAb 进行 CUT&RUN 分析。使用 DNA Library Prep Kit for Illumina (ChIP-seq, CUT&RUN) #56795 制备 DNA 库。该图显示了 Stat1 的已知靶标基因 FZD5 基因内的结合(参见包含 CUT&RUN-qPCR 数据的其他结果图)。
CUT and RUN Image 3: Stat1 (D1K9Y) Rabbit mAb Expand Image
使用 CUT&RUN Assay Kit #86652 对 HT-1080 细胞和 Stat1 (D1K9Y) Rabbit mAb 进行 CUT&RUN 分析。使用 DNA Library Prep Kit for Illumina (ChIP-seq, CUT&RUN) #56795 制备 DNA 库。该图显示了在染色体 2(上图)内的结合,包括 Stat1 的已知靶标基因 FZD5 基因(下图)(参见包含 CUT&RUN-qPCR 数据的其他结果图)。