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.

使用に関する制限

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Revision 7
#12041

Glucocorticoid Receptor (D6H2L) XP® Rabbit mAb

Western Blotting Image 1: Glucocorticoid Receptor (D6H2L) XP® Rabbit mAb Expand Image
Western blot analysis of extracts from various cell lines using Glucocorticoid Receptor (D6H2L) XP® Rabbit mAb.
Western Blotting Image 2: Glucocorticoid Receptor (D6H2L) XP® Rabbit mAb Expand Image
Western blot analysis of extracts from 293T cells, either mock transfected (-) or transfected with constructs expressing Myc/DDK-tagged full-length human glucocorticoid receptor-α (hGRα-Myc/DDK; +) or Myc/DDK-tagged full-length human mineralocorticoid receptor (hMR-Myc/DDK; +), using Glucocorticoid Receptor (D6H2L) XP® Rabbit mAb (upper) or DYKDDDDK Tag Antibody #2368 (lower).
Western Blotting Image 3: Glucocorticoid Receptor (D6H2L) XP® Rabbit mAb Expand Image
Western blot analysis of extracts from control HeLa cells (lane 1), or Glucocorticoid Receptor knockout HeLa cells (lane 2) using Glucocorticoid Receptor (D6H2L) XP® Rabbit mAb #12041 (upper) or GAPDH (D16H11) XP® Rabbit mAb #5174 (lower). The absence of signal in Glucocorticoid Receptor-knockout HeLa cells confirms specificity of the antibody for Glucocorticoid Receptor.
Immunoprecipitation Image 1: Glucocorticoid Receptor (D6H2L) XP® Rabbit mAb Expand Image
Immunoprecipitation of glucocorticoid receptor from HeLa cell extracts using Rabbit (DA1E) mAb IgG XP® Isotype Control #3900 (lane 2) or Glucocorticoid Receptor (D6H2L) XP® Rabbit mAb (lane 3). Lane 1 is 10% input. Western blot analysis was performed using Glucocorticoid Receptor (D6H2L) XP® Rabbit mAb.
Immunohistochemistry Image 1: Glucocorticoid Receptor (D6H2L) XP® Rabbit mAb Expand Image
Immunohistochemical analysis of paraffin-embedded human colon carcinoma using Glucocorticoid Receptor (D6H2L) XP® Rabbit mAb.
Immunohistochemistry Image 2: Glucocorticoid Receptor (D6H2L) XP® Rabbit mAb Expand Image
Immunohistochemical analysis of paraffin-embedded human lung carcinoma using Glucocorticoid Receptor (D6H2L) XP® Rabbit mAb.
Immunohistochemistry Image 3: Glucocorticoid Receptor (D6H2L) XP® Rabbit mAb Expand Image
Immunohistochemical analysis of paraffin-embedded mouse stomach using Glucocorticoid Receptor (D6H2L) XP® Rabbit mAb.
Immunohistochemistry Image 4: Glucocorticoid Receptor (D6H2L) XP® Rabbit mAb Expand Image
Immunohistochemical analysis of paraffin-embedded HeLa cell pellets, untreated (left) or dexamethasone-treated (right), using Glucocorticoid Receptor (D6H2L) XP® Rabbit mAb.
Immunohistochemistry Image 5: Glucocorticoid Receptor (D6H2L) XP® Rabbit mAb Expand Image
Immunohistochemical analysis of paraffin-embedded human prostate carcinoma using Glucocorticoid Receptor (D6H2L) XP® Rabbit mAb.
Immunofluorescence Image 1: Glucocorticoid Receptor (D6H2L) XP® Rabbit mAb Expand Image
Confocal immunofluorescent analysis of HeLa cells (left, positive) or HT-29 cells (right, negative) using Glucocorticoid Receptor (D6H2L) XP® Rabbit mAb #12041 (green), DyLight 650 Phalloidin #12956 (red), and DAPI #4083 (blue).
Immunofluorescence Image 2: Glucocorticoid Receptor (D6H2L) XP® Rabbit mAb Expand Image
Confocal immunofluorescent analysis of HeLa cells (left, positive) or HT-29 cells (right, negative) using Glucocorticoid Receptor (D6H2L) XP® Rabbit mAb #12041 (green), DyLight 650 Phalloidin #12956 (red), and DAPI #4083 (blue).
Flow Cytometry Image 1: Glucocorticoid Receptor (D6H2L) XP® Rabbit mAb Expand Image
Flow cytometric analysis of HT-29 cells (blue, negative) and HeLa cells (green, positive) using Glucocorticoid Receptor (D6H2L) XP® Rabbit mAb (solid lines) or a 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 was used as a secondary antibody.
Flow Cytometry Image 2: Glucocorticoid Receptor (D6H2L) XP® Rabbit mAb Expand Image
Human whole blood was fixed, lysed, and permeabilized as per the Cell Signaling Technology Flow Cytometry (Alternate) Protocol, and stained with CD3-PE, CD19-APC, and Glucocorticoid Receptor (D6H2L) XP® Rabbit mAb. B cell (green) and T cell (blue) population gates (left) were applied to a histogram depicting the mean fluorescence intensity of glucocorticoid receptor, compared to a nonspecific negative control antibody (red; right). Anti-rabbit IgG (H+L), F(ab')2 Fragment (Alexa Fluor® 488 Conjugate) #4412 was used as a secondary antibody.
Chromatin Immunoprecipitation Image 1: Glucocorticoid Receptor (D6H2L) XP® Rabbit mAb Expand Image
Chromatin immunoprecipitations were performed with cross-linked chromatin from A549 cells cultured in media with 5% charcoal-stripped FBS for 3 d and then treated with 100 nM dexamethasone for 1 hr and Glucocorticoid Receptor (D6H2L) XP® Rabbit mAb, using SimpleChIP® Enzymatic Chromatin IP Kit (Magnetic Beads) #9003. DNA Libraries were prepared using DNA Library Prep Kit for Illumina (ChIP-seq, CUT&RUN) #56795. The figure shows binding across SLC19A2, a known target gene of GR (see additional figure containing ChIP-qPCR data). For additional ChIP-seq tracks, please download the product datasheet.
Chromatin Immunoprecipitation Image 2: Glucocorticoid Receptor (D6H2L) XP® Rabbit mAb Expand Image
Chromatin immunoprecipitations were performed with cross-linked chromatin from A549 cells cultured in media with 5% charcoal-stripped FBS for 3 d and then treated with 100 nM dexamethasone for 1 hr and Glucocorticoid Receptor (D6H2L) XP® Rabbit mAb, using SimpleChIP® Enzymatic Chromatin IP Kit (Magnetic Beads) #9003. DNA Libraries were prepared using DNA Library Prep Kit for Illumina (ChIP-seq, CUT&RUN) #56795. The figure shows binding across chromosome 1 (upper), including SLC19A2 (lower), a known target gene of GR (see additional figure containing ChIP-qPCR data).
Chromatin Immunoprecipitation Image 3: Glucocorticoid Receptor (D6H2L) XP® Rabbit mAb Expand Image
A549 cells were cultured in media with 5% charcoal-stripped FBS for 3 d and then either untreated (left panel) or dexamethasone-treated (100 nM, 1 hr; right panel). Chromatin immunoprecipitations were performed with cross-linked chromatin from A549 cells and Glucocorticoid Receptor (D6H2L) XP® 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 SimpleChIP® Human SLC19A2 Promoter Primers #7681, human MT2A 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 1: Glucocorticoid Receptor (D6H2L) XP® Rabbit mAb Expand Image
CUT&RUN was performed with 2.5mg mouse liver tissue fixed with 0.1% formaldehyde for 10 minutes and Glucocorticoid Receptor (D6H2L) XP® 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 8 (upper), including Chd9 (lower), a known target gene of Glucocorticoid Receptor (see additional figure containing CUT&RUN-qPCR data).
CUT and RUN Image 2: Glucocorticoid Receptor (D6H2L) XP® Rabbit mAb Expand Image
CUT&RUN was performed with 2.5mg mouse liver tissue fixed with 0.1% formaldehyde for 10 minutes and Glucocorticoid Receptor (D6H2L) XP® 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 figure shows binding across Chd9, a known target gene of Glucocorticoid Receptor (see additional figure containing CUT&RUN-qPCR data).
CUT and RUN Image 3: Glucocorticoid Receptor (D6H2L) XP® Rabbit mAb Expand Image
CUT&RUN was performed with 2.5 mg mouse liver tissue fixed with 0.1% formaldehyde for 10 minutes and either Glucocorticoid Receptor (D6H2L) XP® 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 mouse A230028O05Rik promoter primers, mouse Chd9 Intron 2 primers and SimpleChIP® Mouse Intracisternal A-Particle (IAP) LTR Primers #85916. 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.