Revision 7
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, IHC-P, IF-IC, FC-FP, ChIP, ChIP-seq, C&R

REACTIVITY:

H M R Mk

SENSITIVITY:

Endogenous

MW (kDa):

94, 91

Source/Isotype:

Rabbit IgG

UniProt ID:

#P04150

Entrez-Gene Id:

2908

Product Information

Product Usage Information

For optimal ChIP and ChIP-seq 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
Immunoprecipitation 1:100
Immunohistochemistry (Paraffin) 1:200 - 1:800
Immunofluorescence (Immunocytochemistry) 1:800 - 1:1600
Flow Cytometry (Fixed/Permeabilized) 1:50 - 1:200
Chromatin IP 1:50
Chromatin IP-seq 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.

Specificity / Sensitivity

Glucocorticoid Receptor (D6H2L) XP® Rabbit mAb recognizes endogenous levels of total GR protein. This antibody reacts with GR-α and GR-β but does not cross-react with mineralocorticoid receptor.

Species Reactivity:

Human, Mouse, Rat, Monkey

Source / Purification

Monoclonal antibody is produced by immunizing animals with a recombinant protein specific to the amino terminus of human GR protein.

Background

Glucocorticoid hormones control cellular proliferation, inflammation, and metabolism through their association with the glucocorticoid receptor (GR)/NR3C1, a member of the nuclear hormone receptor superfamily of transcription factors (1). GR is composed of several conserved structural elements, including a carboxy-terminal ligand-binding domain (which also contains residues critical for receptor dimerization and hormone-dependent gene transactivation), a neighboring hinge region containing nuclear localization signals, a central zinc-finger-containing DNA-binding domain, and an amino-terminal variable region that participates in ligand-independent gene transcription. In the absence of hormone, a significant population of GR is localized to the cytoplasm in an inactive form via its association with regulatory chaperone proteins, such as HSP90, HSP70, and FKBP52. On hormone binding, GR is released from the chaperone complex and translocates to the nucleus as a dimer to associate with specific DNA sequences termed glucocorticoid response elements (GREs), thereby enhancing or repressing transcription of specific target genes (2). It was demonstrated that GR-mediated transcriptional activation is modulated by phosphorylation (3-5). Although GR can be basally phosphorylated in the absence of hormone, it becomes hyperphosphorylated upon binding receptor agonists. It has been suggested that hormone-dependent phosphorylation of GR may determine target promoter specificity, cofactor interaction, strength and duration of receptor signaling, receptor stability, and receptor subcellular localization (3).

  1. Yamamoto, K.R. (1985) Annu. Rev. Genet 19, 209-52.
  2. Necela, B.M. and Cidlowski, J.A. (2003) Trends Pharmacol. Sci. 24, 58-61.
  3. Wang, Z. et al. (2002) J. Biol. Chem. 277, 26573-80.
  4. Rogatsky, I. et al. (1998) J. Biol. Chem. 273, 14315-21.
  5. Krstic, M. D. et al. (1997) Mol. Cell. Biol. 17, 3947-54.

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 IP: Immunoprecipitation IHC-P: Immunohistochemistry (Paraffin) IF-IC: Immunofluorescence (Immunocytochemistry) FC-FP: Flow Cytometry (Fixed/Permeabilized) ChIP: Chromatin IP ChIP-seq: Chromatin IP-seq 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

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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 7
#12041

Glucocorticoid Receptor (D6H2L) XP® Rabbit mAb

Western Blotting Image 1: Glucocorticoid Receptor (D6H2L) XP® Rabbit mAb Expand Image
使用 Glucocorticoid Receptor (D6H2L) XP® Rabbit mAb,对不同细胞系提取物进行蛋白质印迹分析。
Western Blotting Image 2: Glucocorticoid Receptor (D6H2L) XP® Rabbit mAb Expand Image
使用 Glucocorticoid Receptor (D6H2L) XP® Rabbit mAb(上)或 DYKDDDDK Tag Antibody #2368(下),对转染空载 (-) 或转染带有 Myc/DDK 标签的全长人糖皮质激素受体 α (hGRα-Myc/DDK; +)、带有Myc/DDK 标签的全长人盐皮质激素受体 (hMR-Myc/DDK; +) 表达载体的 293T 细胞提取物进行蛋白质印迹分析。
Western Blotting Image 3: Glucocorticoid Receptor (D6H2L) XP® Rabbit mAb Expand Image
使用 Glucocorticoid Receptor (D6H2L) XP® Rabbit mAb #12041(上图)或 GAPDH (D16H11) XP® Rabbit mAb #5174(下图)对对照 HeLa 细胞(泳道 1)或糖皮质激素受体敲除型 HeLa 细胞(泳道 2)的提取物进行蛋白质印迹分析。糖皮质激素受体敲除型 HeLa 细胞中没有信号,这证实了抗体对糖皮质激素受体的特异性。
Immunoprecipitation Image 1: Glucocorticoid Receptor (D6H2L) XP® Rabbit mAb Expand Image
使用 Rabbit (DA1E) mAb IgG XP® Isotype Control #3900(泳道 2)或 Glucocorticoid Receptor (D6H2L) XP® Rabbit mAb(泳道 3),对 HeLa 细胞提取物糖皮质激素受体进行免疫沉淀。泳道 1 是 10% 输入对照。使用 Glucocorticoid Receptor (D6H2L) XP® Rabbit mAb 进行蛋白质印迹分析。
Immunohistochemistry Image 1: Glucocorticoid Receptor (D6H2L) XP® Rabbit mAb Expand Image
使用 Glucocorticoid Receptor (D6H2L) XP® Rabbit mAb 对石蜡包埋的人结肠癌进行免疫组织化学分析。
Immunohistochemistry Image 2: Glucocorticoid Receptor (D6H2L) XP® Rabbit mAb Expand Image
使用 Glucocorticoid Receptor (D6H2L) XP® Rabbit mAb 对石蜡包埋的人肺癌进行免疫组织化学分析。
Immunohistochemistry Image 3: Glucocorticoid Receptor (D6H2L) XP® Rabbit mAb Expand Image
使用 Glucocorticoid Receptor (D6H2L) XP® Rabbit mAb 对石蜡包埋的小鼠胃进行免疫组织化学分析。
Immunohistochemistry Image 4: Glucocorticoid Receptor (D6H2L) XP® Rabbit mAb Expand Image
使用 Glucocorticoid Receptor (D6H2L) XP® Rabbit mAb,对未经处理(左)或经地塞米松处理(右)的石蜡包埋 HeLa 细胞沉淀物进行免疫组织化学分析。
Immunohistochemistry Image 5: Glucocorticoid Receptor (D6H2L) XP® Rabbit mAb Expand Image
使用 Glucocorticoid Receptor (D6H2L) XP® Rabbit mAb对石蜡包埋的人前列腺癌进行免疫组织化学分析。
Immunofluorescence Image 1: Glucocorticoid Receptor (D6H2L) XP® Rabbit mAb Expand Image
使用 Glucocorticoid Receptor (D6H2L) XP® Rabbit mAb #12041(绿色)、 DyLight 650 Phalloidin #12956(红色)和 DAPI #4083(蓝色)对 HeLa 细胞(左图,阳性)或 HT-29 细胞(右图,阴性)进行共聚焦免疫荧光分析。
Immunofluorescence Image 2: Glucocorticoid Receptor (D6H2L) XP® Rabbit mAb Expand Image
使用 Glucocorticoid Receptor (D6H2L) XP® Rabbit mAb #12041(绿色)、 DyLight 650 Phalloidin #12956(红色)和 DAPI #4083(蓝色)对 HeLa 细胞(左图,阳性)或 HT-29 细胞(右图,阴性)进行共聚焦免疫荧光分析。
Flow Cytometry Image 1: Glucocorticoid Receptor (D6H2L) XP® Rabbit mAb Expand Image
使用 Glucocorticoid Receptor (D6H2L) XP® Rabbit mAb(实线)或浓度匹配的 Rabbit (DA1E) mAb IgG XP® Isotype Control #3900(虚线)对 HT-29 细胞(蓝色,阴性)和 HeLa 细胞(绿色,阳性)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。
Flow Cytometry Image 2: Glucocorticoid Receptor (D6H2L) XP® Rabbit mAb Expand Image
按照 Cell Signaling Technology 流式细胞术(备选)实验步骤对人全血进行固定、溶解和通透处理,并用 CD3-PE、CD19-APC 和 Glucocorticoid Receptor (D6H2L) XP® Rabbit mAb 染色。B 细胞(绿色)和 T 细胞(蓝色)群门道(左),适用于描绘与非特异性阴性对照抗体(红色;右)相对比的糖皮质激素受体的平均荧光强度的柱状图。Anti-rabbit IgG (H+L)、F(ab')2 Fragment (Alexa Fluor® 488 Conjugate) #4412 作为二抗。
Chromatin Immunoprecipitation Image 1: Glucocorticoid Receptor (D6H2L) XP® Rabbit mAb Expand Image
使用 SimpleChIP® Enzymatic Chromatin IP Kit (Magnetic Beads) #9003,对在含有 5% 活性炭剥离 FBS 的培养基中培养 3 天,然后经 100 nM Dexamethasone 处理 1 小时的 A549 细胞中提取的交联染色质,在加入 Glucocorticoid Receptor (D6H2L) XP® Rabbit mAb 后,进行染色质免疫沉淀分析。使用 DNA Library Prep Kit for Illumina (ChIP-seq, CUT&RUN) #56795 制备 DNA 库。本图显示在 SLC19A2 内结合,SLC19A2 是一种已知的 GR 靶标基因(参见包含 ChIP-qPCR 数据的其他图形)。欲知其他 ChIP-seq 情况,请下载产品说明书。
Chromatin Immunoprecipitation Image 2: Glucocorticoid Receptor (D6H2L) XP® Rabbit mAb Expand Image
使用 SimpleChIP® Enzymatic Chromatin IP Kit (Magnetic Beads) #9003,对在含有 5% 活性炭剥离 FBS 的培养基中培养 3 天,然后经 100 nM Dexamethasone 处理 1 小时的 A549 细胞中提取的交联染色质,在加入 Glucocorticoid Receptor (D6H2L) XP® Rabbit mAb 后,进行染色质免疫沉淀分析。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 (上图)内的结合,包括 GR 的已知靶基因 SLC19A2(下图)(参见包含 ChIP-qPCR 数据的其他结果图)。
Chromatin Immunoprecipitation Image 3: Glucocorticoid Receptor (D6H2L) XP® Rabbit mAb Expand Image
A549 细胞在含有 5% 活性炭剥离 FBS 的培养基中培养 3 天,随后不进行处理(左小图)或用塞米松处理(100 nM,1 小时;右小图)。使用 SimpleChIP® Enzymatic Chromatin IP Kit (Magnetic Beads) #9003,对 A549 细胞中提取的交联染色质,在加入 Glucocorticoid Receptor (D6H2L) XP® Rabbit mAb 或 Normal Rabbit IgG #2729 后进行染色质免疫沉淀。使用 SimpleChIP® Human SLC19A2 Promoter Primers #7681、human MT2A promoter primers 和 SimpleChIP® Human α Satellite Repeat Primers #4486,并通过实时 PCR 对富集的 DNA 进行定量。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: Glucocorticoid Receptor (D6H2L) XP® Rabbit mAb Expand Image
使用 CUT&RUN Assay Kit #86652 ,以采用 0.1% 甲醛固定 10 分钟的 2.5mg 小鼠肝组织和 Glucocorticoid Receptor (D6H2L) XP® Rabbit mAb 进行 CUT&RUN 。使用 DNA Library Prep Kit for Illumina (ChIP-seq, CUT&RUN) #56795 制备 DNA 库。这些结果图显示跨整个染色体 8(上图)的结合,包括糖皮质激素受体的已知靶基因 Chd9(下图)(参见包含 ChIP-qPCR 数据的其他结果图)。
CUT and RUN Image 2: Glucocorticoid Receptor (D6H2L) XP® Rabbit mAb Expand Image
使用 CUT&RUN Assay Kit #86652 ,以采用 0.1% 甲醛固定 10 分钟的 2.5mg 小鼠肝组织和 Glucocorticoid Receptor (D6H2L) XP® Rabbit mAb 进行 CUT&RUN 。使用 DNA Library Prep Kit for Illumina (ChIP-seq, CUT&RUN) #56795 制备 DNA 库。该结果图显示了跨整个糖皮质激素受体的已知靶基因 Chd9 的结合(参见包含 CUT&RUN-qPCR 数据的其他结果图)。
CUT and RUN Image 3: Glucocorticoid Receptor (D6H2L) XP® Rabbit mAb Expand Image
使用 CUT&RUN Assay Kit #86652 ,以采用0.1% 甲醛固定 10 分钟的 2.5 mg 小鼠肝组织和 Glucocorticoid Receptor (D6H2L) XP® Rabbit mAb 或 Rabbit (DA1E) mAb IgG XP® Isotype Control (CUT&RUN) #66362 进行 CUT&RUN。使用小鼠 A230028O05Rik 启动子引物、小鼠 Chd9 Intron 2 引物和 SimpleChIP® Mouse Intracisternal A-Particle (IAP) LTR Primers #85916,通过实时 PCR 对富集的 DNA 定量。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。