Revision 5

#12721Store 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, IP, IF-IC, FC-FP, ChIP, ChIP-seq

REACTIVITY:

H M Mk

SENSITIVITY:

Endogenous

MW (kDa):

97-100

Source/Isotype:

Rabbit IgG

UniProt ID:

#Q16236

Entrez-Gene Id:

4780

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
Immunoprecipitation 1:50
Immunofluorescence (Immunocytochemistry) 1:200 - 1:800
Flow Cytometry (Fixed/Permeabilized) 1:1600 - 1:6400
Chromatin IP 1:200
Chromatin IP-seq 1:200

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 #84599.

Specificity / Sensitivity

NRF2 (D1Z9C) XP® Rabbit mAb recognizes endogenous levels of total NRF2 protein.

Species Reactivity:

Human, Mouse, Monkey

Source / Purification

Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Ala275 of human NRF2 protein.

Background

The nuclear factor-like 2 (NRF2) transcriptional activator binds antioxidant response elements (ARE) of target gene promoter regions to regulate expression of oxidative stress response genes. Under basal conditions, the NRF2 inhibitor INrf2 (also called KEAP1) binds and retains NRF2 in the cytoplasm where it can be targeted for ubiquitin-mediated degradation (1). Small amounts of constitutive nuclear NRF2 maintain cellular homeostasis through regulation of basal expression of antioxidant response genes. Following oxidative or electrophilic stress, KEAP1 releases NRF2, thereby allowing the activator to translocate to the nucleus and bind to ARE-containing genes (2). The coordinated action of NRF2 and other transcription factors mediates the response to oxidative stress (3). Altered expression of NRF2 is associated with chronic obstructive pulmonary disease (COPD) (4). NRF2 activity in lung cancer cell lines directly correlates with cell proliferation rates, and inhibition of NRF2 expression by siRNA enhances anti-cancer drug-induced apoptosis (5).

  1. Cullinan, S.B. et al. (2004) Mol Cell Biol 24, 8477-86.
  2. Nguyen, T. et al. (2005) J Biol Chem 280, 32485-92.
  3. Jaiswal, A.K. (2004) Free Radic Biol Med 36, 1199-207.
  4. Suzuki, M. et al. (2008) Am J Respir Cell Mol Biol 39, 673-82.
  5. Homma, S. et al. (2009) Clin Cancer Res 15, 3423-32.

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 nonfat dry milk, 1X TBS, 0.1% Tween® 20 at 4°C with gentle shaking, overnight.

Applications Key

WB: Western Blotting IP: Immunoprecipitation 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.
XP is a registered 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 5
#12721

NRF2 (D1Z9C) XP® Rabbit mAb

Western Blotting Image 1: NRF2 (D1Z9C) XP® Rabbit mAb Expand Image
使用 NRF2 (D1Z9C) XP® Rabbit mAb 对未经处理 (-) 或经 MG-132 #2194(10 μM,10 小时;+)处理的 MEF wt 和 U-2 OS 细胞的提取物进行蛋白质印迹分析。
Immunoprecipitation Image 1: NRF2 (D1Z9C) XP® Rabbit mAb Expand Image
使用 Rabbit (DA1E) mAb IgG XP® Isotype Control #3900(泳道 2)或 NRF2 (D1Z9C) XP® Rabbit mAb(泳道 3),对经 MG-132 #2194 处理(10 μM,10 小时)的 MEF wt 细胞提取物 NRF2 进行免疫沉淀。泳道 1 是 10% 输入对照。使用 NRF2 (D1Z9C) XP® Rabbit mAb(泳道 3) 进行蛋白质印迹分析。
Immunofluorescence Image 1: NRF2 (D1Z9C) XP® Rabbit mAb Expand Image
使用 NRF2 (D1Z9C) XP® Rabbit mAb(绿色伪彩)对未经处理(左图)或已经过 MG-132 #2194(10 μM,8 小时;中图)处理的野生型 NRF2 MEF 细胞和已经过 MG-132 #2194 (10 μM,8 小时,右图)处理的 NRF2 敲除型 MEF 细胞进行共聚焦免疫荧光分析。肌动蛋白丝用 Alexa Fluor® 488 Phalloidin #8878(红色伪彩)标记。
Flow Cytometry Image 1: NRF2 (D1Z9C) XP® Rabbit mAb Expand Image
使用 NRF2 (D1Z9C) XP® 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)或经过 MG-132 #2194(10 uM,4 小时;绿色)处理的 MEF wt 细胞进行流式细胞分析。Anti-rabbit IgG (H+L), F(ab')2 Fragment (Alexa Fluor® 488 Conjugate) #4412 用作二抗。
Chromatin Immunoprecipitation Image 1: NRF2 (D1Z9C) XP® Rabbit mAb Expand Image
使用 SimpleChIP® 酶解染色质免疫沉淀试剂盒(磁珠法) #9005 对经 DEM(50 μM,3 小时)处理的 MEF 细胞的交联染色质与 NRF2 (D1Z9C) XP® 兔单克隆抗体进行染色质免疫沉淀。使用 DNA Library Prep Kit for Illumina Systems (ChIP-seq, CUT&RUN) #56795 制备 DNA 库。结果图显示在 NRF2 的已知靶基因 NQO1 内结合(参见包含 ChIP-qPCR 数据的其他结果图)。
Chromatin Immunoprecipitation Image 2: NRF2 (D1Z9C) XP® Rabbit mAb Expand Image
使用 SimpleChIP® 酶解染色质免疫沉淀试剂盒(磁珠法) #9005 对经 DEM(50 μM,3 小时)处理的 MEF 细胞的交联染色质与 NRF2 (D1Z9C) XP® 兔单克隆抗体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8(上图)内的结合,包括 NRF2 的已知靶基因 NQO1(下图)(参见包含 ChIP-qPCR 数据的其他结果图)。
Chromatin Immunoprecipitation Image 3: NRF2 (D1Z9C) XP® Rabbit mAb Expand Image
使用 SimpleChIP® Enzymatic Chromatin IP Kit (Magnetic Beads) #9003,对已经 DEM 处理(50 μM,3 小时)的 MEF NRF2 野生型(左图)和 NRF2 敲除型(右图)细胞中提取的交联染色质,在加入 NRF2 (D1Z9C) XP® Rabbit mAb 或 Normal Rabbit IgG #2729 后进行染色质免疫沉淀。使用 mouse MafG intron 1 primers、SimpleChIP® Mouse NQO1 Promoter Primers #12635 和 SimpleChIP® Mouse RPL30 Intron 2 Primers #7015,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。