Recombinant Anti-Glucocorticoid receptor antibody [EPR24889-231] (ab305050)
Key features and details
- Produced recombinantly (animal-free) for high batch-to-batch consistency and long term security of supply
- Rabbit monoclonal [EPR24889-231] to Glucocorticoid Receptor
- Suitable for: ChIP, IP, ICC/IF, WB, IHC-P
- Knockout validated
- Reacts with: Human
Related conjugates and formulations
Overview
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Product name
Anti-Glucocorticoid receptor antibody [EPR24889-231]
See all Glucocorticoid Receptor primary antibodies -
Description
Rabbit monoclonal [EPR24889-231] to Glucocorticoid Receptor -
Host species
Rabbit -
Tested applications
Suitable for: ChIP, IP, ICC/IF, WB, IHC-Pmore details
Unsuitable for: Flow Cyt (Intra) -
Species reactivity
Reacts with: Human
Does not react with: Mouse, Rat -
Immunogen
Recombinant fragment. This information is proprietary to Abcam and/or its suppliers.
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Positive control
- WB: Whole cell lysates: HeLa (human cervix adenocarcinoma epithelial cell), Glucocorticoid receptor knockout HeLa, U-87 MG (human glioblastoma-astrocytoma epithelial cell), HEK-293T (human embryonic kidney epithelial cell), THP-1 (human monocytic leukemia monocyte). IHC-P: Human: liver, pancreas, and kidney. ICC/IF: NR3C1 knockout human cervical adenocarcinoma epithelial cell. IP: HeLa. ChIP: A549(Human lung carcinoma epithelial cell) treated with dexamethasone(100nM 1h) and A549 non treated.
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General notes
This product is a recombinant monoclonal antibody, which offers several advantages including:
- - High batch-to-batch consistency and reproducibility
- - Improved sensitivity and specificity
- - Long-term security of supply
- - Animal-free production
Our RabMAb® technology is a patented hybridoma-based technology for making rabbit monoclonal antibodies. For details on our patents, please refer to RabMAb® patents.
Properties
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Form
Liquid -
Storage instructions
Shipped at 4°C. Store at +4°C short term (1-2 weeks). Upon delivery aliquot. Store at -20°C long term. Avoid freeze / thaw cycle. -
Storage buffer
pH: 7.2
Preservative: 0.01% Sodium azide
Constituents: 59% PBS, 40% Glycerol (glycerin, glycerine), 0.05% BSA -
Concentration information loading...
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Purity
Protein A purified -
Clonality
Monoclonal -
Clone number
EPR24889-231 -
Isotype
IgG -
Research areas
Associated products
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Alternative Versions
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Compatible Secondaries
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Isotype control
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KO cell lines
Applications
The Abpromise guarantee
Our Abpromise guarantee covers the use of ab305050 in the following tested applications.
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
Application | Abreviews | Notes |
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ChIP |
Use a concentration of 5 µg/ml.
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IP |
1/30.
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ICC/IF |
1/50.
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WB |
1/1000. Detects a band of approximately 94 kDa (predicted molecular weight: 85 kDa).
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IHC-P |
1/100. Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.
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Notes |
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ChIP
Use a concentration of 5 µg/ml. |
IP
1/30. |
ICC/IF
1/50. |
WB
1/1000. Detects a band of approximately 94 kDa (predicted molecular weight: 85 kDa). |
IHC-P
1/100. Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol. |
Target
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Function
Receptor for glucocorticoids (GC). Has a dual mode of action: as a transcription factor that binds to glucocorticoid response elements (GRE) and as a modulator of other transcription factors. Affects inflammatory responses, cellular proliferation and differentiation in target tissues. Could act as a coactivator for STAT5-dependent transcription upon growth hormone (GH) stimulation and could reveal an essential role of hepatic GR in the control of body growth. Involved in chromatin remodeling. Plays a significant role in transactivation. Involved in nuclear translocation. -
Tissue specificity
Widely expressed. In the heart, detected in left and right atria, left and right ventricles, aorta, apex, intraventricular septum, and atrioventricular node as well as whole adult and fetal heart. -
Involvement in disease
Defects in NR3C1 are a cause of glucocorticoid resistance (GCRES) [MIM:138040]; also known as cortisol resistance. It is a hypertensive, hyperandrogenic disorder characterized by increased serum cortisol concentrations. Inheritance is autosomal dominant. -
Sequence similarities
Belongs to the nuclear hormone receptor family. NR3 subfamily.
Contains 1 nuclear receptor DNA-binding domain. -
Domain
Composed of three domains: a modulating N-terminal domain, a DNA-binding domain and a C-terminal ligand-binding domain. -
Post-translational
modificationsIncreased proteasome-mediated degradation in response to glucocorticoids.
Phosphorylated in the absence of hormone; becomes hyperphosphorylated in the presence of glucocorticoid. The Ser-203-phosphorylated form is mainly cytoplasmic, and the Ser-211-phosphorylated form is nuclear. Transcriptional activity correlates with the amount of phosphorylation at Ser-211.
Sumoylated; this reduces transcription transactivation.
Ubiquitinated; restricts glucocorticoid-mediated transcriptional signaling. -
Cellular localization
Cytoplasm. Nucleus. Cytoplasmic in the absence of ligand, nuclear after ligand-binding and Nucleus. Localized largely in the nucleus. - Information by UniProt
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Database links
- Entrez Gene: 2908 Human
- Omim: 138040 Human
- SwissProt: P04150 Human
- Unigene: 122926 Human
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Alternative names
- GCCR antibody
- GCR antibody
- GCR_HUMAN antibody
see all
Images
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All lanes : Anti-Glucocorticoid receptor antibody [EPR24889-231] (ab305050) at 1/1000 dilution
Lane 1 : Wild-type HeLa (human cervix adenocarcinoma epithelial cell), whole cell lysate
Lane 2 : Glucocorticoid receptor knockout HeLa (human cervix adenocarcinoma epithelial cell), whole cell lysate
Lane 3 : U-87 MG (human glioblastoma-astrocytoma epithelial cell), whole cell lysate
Lysates/proteins at 20 µg per lane.
Secondary
All lanes : Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (ab216773) at 1/10000 dilution
Predicted band size: 85 kDa
Observed band size: 94 kDa why is the actual band size different from the predicted?Blocking and diluting buffer and concentration: Intercept® (TBS) Blocking Buffer diluted with an equal volume of 0.1% TBS.
The bands beneath the target band (94 kDa) are likely to be degraded target fragments.
False colour image of Western blot: Anti-Glucocorticoid receptor antibody [EPR24889-231] (ab305050) staining at 1/1000 dilution, shown in green; Mouse anti-GAPDH antibody [6C5] (ab8245) loading control staining at 1/20000 dilution, shown in red.
In Western blot, ab305050 was shown to bind specifically to Glucocorticoid receptor. A band was observed at 94 kDa in wild-type HeLa cell lysates with no signal observed at this size in Glucocorticoid receptor knockout cell line. To generate this image, wild-type and Glucocorticoid receptor knockout HeLa cell lysates were analysed. First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 3 % milk in TBS-0.1 % Tween® 20 (TBS-T) before incubation with primary antibodies overnight at 4 °C. Blots were washed four times in TBS-T, incubated with secondary antibodies for 1 h at room temperature, washed again four times then imaged. Secondary antibodies used were Goat anti-Rabbit IgG H&L (IRDye® 800CW) preabsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye® 680RD) preabsorbed (ab216776) at 1/10000 dilution. -
All lanes : Anti-Glucocorticoid receptor antibody [EPR24889-231] (ab305050) at 1/1000 dilution
Lane 1 : HeLa (human cervix adenocarcinoma epithelial cell), whole cell lysate
Lane 2 : HEK-293T (human embryonic kidney epithelial cell), whole cell lysate
Lane 3 : THP-1 (human monocytic leukemia monocyte), whole cell lysate
Lysates/proteins at 20 µg per lane.
Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution
Performed under non-reducing conditions.
Predicted band size: 85 kDa
Observed band size: 94 kDa why is the actual band size different from the predicted?Blocking and diluting buffer and concentration: 5% NFDM/TBST.
The bands beneath the target band (94 kDa) in lane 3 are likely to be degraded target fragments.
Lysates were freshly made and used immediately to minimize protein degradation.Exposure time: Lane 1: 37 seconds
Lanes 2-3: 3 minutes -
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Glucocorticoid receptor antibody [EPR24889-231] to (ab305050)
Immunohistochemical analysis of paraffin-embedded human liver tissue labeling Glucocorticoid receptor with ab305050 at 1/100 dilution (5.12 µg/ml), followed by a ready to use Leica DS9800 (Bond™ Polymer Refine Detection). Nuclear staining on human liver is observed. The section was incubated with ab305050 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND® RX instrument. Counterstained with Hematoxylin.
Secondary antibody only control: Secondary antibody is a ready to use Leica DS9800 (Bond™ Polymer Refine Detection) kit.
Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope retrieval solution 2) for 20 mins.
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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Glucocorticoid receptor antibody [EPR24889-231] to (ab305050)
Immunohistochemical analysis of paraffin-embedded human pancreas tissue labeling Glucocorticoid receptor with ab305050 at 1/100 dilution (5.12 µg/ml), followed by a ready to use Leica DS9800 (Bond™ Polymer Refine Detection). Nuclear staining on human pancreas is observed (PMID: 32619553). The section was incubated with ab305050 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND® RX instrument. Counterstained with Hematoxylin.
Secondary antibody only control: Secondary antibody is a ready to use Leica DS9800 (Bond™ Polymer Refine Detection) kit.
Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope retrieval solution 2) for 20 mins.
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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Glucocorticoid receptor antibody [EPR24889-231] to (ab305050)
Immunohistochemical analysis of paraffin-embedded human kidney tissue labeling Glucocorticoid receptor with ab305050 at 1/100 dilution (5.12 µg/ml), followed by a ready to use Leica DS9800 (Bond™ Polymer Refine Detection). Nuclear staining on human kidney is observed. The section was incubated with ab305050 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND® RX instrument. Counterstained with Hematoxylin.
Secondary antibody only control: Secondary antibody is a ready to use Leica DS9800 (Bond™ Polymer Refine Detection) kit.
Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope retrieval solution 2) for 20 mins.
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Immunocytochemistry/ Immunofluorescence - Anti-Glucocorticoid receptor antibody [EPR24889-231] to (ab305050)
Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized NR3C1 KO HeLa (NR3C1 knockout human cervical adenocarcinoma epithelial cell) (ab261766) cells labeling Glucocorticoid receptor with AB305050 at 1/50 dilution (10.24 µg/mL), followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed antibody at 1/1000 dilution (2 µg/mL) (Green). Confocal image showing no staining in NR3C1 knockout HeLa cells and showing nuclear and weak cytoplasmic staining in wildtype HeLa cells. The image was taken with a confocal microscope (Leica-Microsystems, TCS SP8). ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor® 594) was used to counterstain tubulin at 1/200 (2.5 µg/mL) (Red). The nuclear counterstain was DAPI (Blue).
Secondary antibody only control: Secondary antibody is ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1000 dilution (2 µg/mL).
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Glucocorticoid receptor was immunoprecipitated from 0.35 mg HeLa (human cervix adenocarcinoma epithelial cell) whole cell lysate with ab305050 at 1/30 dilution (2 μg in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab305050 at dilution. VeriBlot for IP secondary antibody (HRP) (ab131366) was used at 1/5000 dilution.
Lane 1: HeLa (human cervix adenocarcinoma epithelial cell) whole cell lysate 10 µg (Inset)
Lane 2: ab305050 IP in HeLa whole cell lysate
Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab305050 in HeLa whole cell lysate
Blocking and dilution buffer and concentration: 5% NFDM/TBST.
Exposure time: 6 seconds
Observed MW (kDa): 94.
The bands beneath the target band (94 kDa) are likely to be degraded target fragments.
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Chromatin was prepared from A549+dexamethasone(100 nM 1h) cells according to the Abcam Dual-X-ChIP protocol*. Cells were fixed with 1.5 mM EGS for 30 mins and then formaldehyde for 10 min.
The ChIP was performed with 25 µg of chromatin, 5 µg of 305050 (red), or 5 µg of rabbit normal IgG ab172730 (gray) and 25 µl of Protein A/G Dynabeads.The immunoprecipitated DNA was quantified by real time PCR (Sybr green approach).
*http://www.abcam.com/resources?keywords=X%20ChIP%20protocol
Protocols
To our knowledge, customised protocols are not required for this product. Please try the standard protocols listed below and let us know how you get on.
Datasheets and documents
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SDS download
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Datasheet download
Certificate of Compliance
References (0)
ab305050 has not yet been referenced specifically in any publications.