Overview

  • Product name
    Anti-Glucocorticoid Receptor antibody [BuGR2] - ChIP Grade
    See all Glucocorticoid Receptor primary antibodies
  • Description
    Mouse monoclonal [BuGR2] to Glucocorticoid Receptor - ChIP Grade
  • Host species
    Mouse
  • Specificity
    Immunocytochemical staining of GR in L929 cells with this antibody results in staining of both the cytoplasm and nucleus, even in the presence of hormone. This antibody, using enzymatic digestion analysis, has been shown to react with the undigested 97 kDa GR, a 17 kDa DNA-binding trypsin fragment, and a 45 kDa steroid- and DNA-binding chymotrypsin fragment.
  • Tested applications
    Suitable for: ICC/IF, ChIP, ELISA, IHC-FoFr, Flow Cyt, IHC-P, IP, WBmore details
  • Species reactivity
    Reacts with: Mouse, Rat, Sheep, Rabbit, Guinea pig, Human, Saccharomyces cerevisiae, Xenopus laevis
    Does not react with: Bird, Non human primates, Amphibian
  • Immunogen

    Other Immunogen Type corresponding to Rat Glucocorticoid Receptor. Partially purified rat GR.

  • Positive control
    • ICC: L929 cells WB: Mouse pituitary tumors, mammary tissue, placenta, liver and thymus lysates.

Properties

Applications

Our Abpromise guarantee covers the use of ab2768 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
ICC/IF 1/50 - 1/500.
ChIP Use at an assay dependent concentration.
ELISA Use at an assay dependent concentration.
IHC-FoFr 1/500. PubMed: 20307510
EMSA Use at an assay dependent concentration.
Flow Cyt Use 0.5-1µg for 106 cells.

ab18414 - Mouse monoclonal IgG2a, is suitable for use as an isotype control with this antibody.

IHC-P Use a concentration of 5 µg/ml. We recommend the following for antigen retrieval: Incubate in 10 mM citrate buffer, pH 6.0 in microwave oven for 7 x 2 minutes (2 minutes, stop, 2 minutes, etc.). Then wash with Dulbecco’s PBS 6 x 7 minutes at room temperature. The tissue is now ready to be blocked for the start of IHC.
IP Use at an assay dependent concentration.
WB Use a concentration of 5 µg/ml. Detects a band of approximately 97 kDa (predicted molecular weight: 86 kDa). Using enzymatic digestion analysis detects a band of approximately 97 kDa, a 17 kDa DNA-binding trypsin fragment, and a 45 kDa steroid- and DNA-binding chymotrypsin fragment (predicted molecular weight: 86 kDa).

Target

  • 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
    modifications
    Increased 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
  • Database links
  • Alternative names
    • GCCR antibody
    • GCR antibody
    • GCR_HUMAN antibody
    • GCRST antibody
    • glucocorticoid nuclear receptor variant 1 antibody
    • Glucocorticoid receptor antibody
    • GR antibody
    • GRL antibody
    • Grl1 antibody
    • nr3c1 antibody
    • Nuclear receptor subfamily 3 group C member 1 antibody
    • nuclear receptor subfamily 3, group C, member 1 (glucocorticoid receptor) antibody
    see all

Images

  • Immunocytochemistry/Immunofluorescence analysis of Glucocorticoid Receptor shows staining in A549 cells. Glucocorticoid Receptor (green), F-Actin staining with Phalloidin (red) and nuclei with DAPI (blue) is shown. Cells were grown on chamber slides and fixed with formaldehyde prior to staining. Cells were probed without (control) or with ab2768 (1:100) over night at 4°C, washed with PBS and incubated with a DyLight-488 conjugated secondary antibody. Images were taken at 60X magnification.

  • Immunocytochemistry/Immunofluorescence analysis of Glucocorticoid Receptor shows staining in HeLa cells. Glucocorticoid Receptor (green), F-Actin staining with Phalloidin (red) and nuclei with DAPI (blue) is shown. Cells were grown on chamber slides and fixed with formaldehyde prior to staining. Cells were probed without (control) or with ab2768 (1:100) over night at 4°C, washed with PBS and incubated with a DyLight-488 conjugated secondary antibody. Images were taken at 60X magnification.

  • Immunocytochemistry/Immunofluorescence analysis of Glucocorticoid Receptor shows staining in U251 cells. Glucocorticoid Receptor (green), F-Actin staining with Phalloidin (red) and nuclei with DAPI (blue) is shown. Cells were grown on chamber slides and fixed with formaldehyde prior to staining. Cells were probed without (control) or with ab2768 (1:100) over night at 4°C, washed with PBS and incubated with a DyLight-488 conjugated secondary antibody. Images were taken at 60X magnification.

  • Flow cytometry analysis of Glucocorticoid Receptor showing positive staining in the nucleus and cytoplasm of NIH/3T3 cells compared to an isotype control (blue). Cells were harvested and adjusted to a concentration of 1-5x10^6 cells/ml. Cells were then fixed with 2% paraformaldehyde and washed with PBS. Cells were penetrated by dropping the supernatant and adding 90% methanol followed by incubation for 10 minutes at room temperature. Cells were blocked with a 2% solution of BSA-PBS for 30 min at room temperature and incubated with ab2768 at 2 ug/test for 60 min at room temperature. Cells were then incubated for 40 min at room temperature in the dark using a Dylight 488-conjugated goat anti-mouse IgG (H+L) secondary antibody and re-suspended in PBS for FACS analysis.
  • Flow cytometry analysis of Glucocorticoid Receptor showing positive staining in the nucleus and cytoplasm of Jurkat cells compared to an isotype control (blue). Cells were harvested and adjusted to a concentration of 1-5x10^6 cells/ml. Cells were then fixed with 2% paraformaldehyde and washed with PBS. Cells were penetrated by dropping the supernatant and adding 90% methanol followed by incubation for 10 minutes at room temperature. Cells were blocked with a 2% solution of BSA-PBS for 30 min at room temperature and then incubated with ab2768 at 1 ug/test for 60 min at room temperature. Cells were then incubated for 40 min at room temperature in the dark using a Dylight 488-conjugated goat anti-mouse IgG (H+L) secondary antibody and re-suspended in PBS for FACS analysis.
  • Flow cytometry analysis of Glucocorticoid Receptor showing positive staining in the nucleus and cytoplasm of Hela cells compared to an isotype control (blue). Cells were harvested and adjusted to a concentration of 1-5x10^6 cells/ml. Cells were then fixed with 2% paraformaldehyde and washed with PBS. Cells were penetrated by dropping the supernatant and adding 90% methanol followed by incubation for 10 minutes at room temperature. Cells were blocked with a 2% solution of BSA-PBS for 30 min at room temperature and incubated with ab2768 at 1 ug/test for 60 min at room temperature. Cells were then incubated for 40 min at room temperature in the dark using a Dylight 488-conjugated goat anti-mouse IgG (H+L) secondary antibody and re-suspended in PBS for FACS analysis.
  • Western blot of glucocorticoid receptor on mouse liver extract using ab2768. Western blot of glucocorticoid receptor on mouse liver extract using ab2768.
  • ICC/IF image of ab2768 stained HepG2 cells. The cells were 100% methanol fixed (5 min) and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab2768, 1µg/ml) overnight at +4°C. The secondary antibody (green) was Alexa Fluor® 488 goat anti-mouse IgG (H+L) used at a 1/1000 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (red) at a 1/200 dilution for 1h. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43µM.

  • Overlay histogram showing Jurkat cells stained with ab2768 (red line). The cells were fixed with 80% methanol (5 min) and then permeabilized with 0.1% PBS-Tween for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (ab2768, 0.5µg/1x106 cells) for 30 min at 22ºC. The secondary antibody used was DyLight® 488 goat anti-mouse IgG (H+L) (ab96879) at 1/500 dilution for 30 min at 22ºC. Isotype control antibody (black line) was mouse IgG2 (1µg/1x106 cells) used under the same conditions. Acquisition of >5,000 events was performed. This antibody gave a positive signal in Jurkat cells fixed with 4% paraformaldehyde (10 min)/permeabilized with 0.1% PBS-Tween for 20 min used under the same conditions.

  • Immunohistochemical analysis of rat brain tissue, staining Glucocorticoid Receptor with ab2768.

    Tissue was fixed with formalin, permeabilized with 0.3% Triton X-100 and blocked with 3% BSA for 1 hour at room temperature; antigen retrieval was via an enzymatic method. Samples were incubated with primary antibody (1/100) for 12 hours at 4°C. A biotinylated goat anti-mouse polyclonal IgG (1/200) was used as the secondary antibody.

    See Abreview

References

This product has been referenced in:
  • Han QQ  et al. Differential GR Expression and Translocation in the Hippocampus Mediates Susceptibility vs. Resilience to Chronic Social Defeat Stress. Front Neurosci 11:287 (2017). Read more (PubMed: 28588443) »
  • Ghnenis AB  et al. Maternal obesity in the ewe increases cardiac ventricular expression of glucocorticoid receptors, proinflammatory cytokines and fibrosis in adult male offspring. PLoS One 12:e0189977 (2017). Read more (PubMed: 29267325) »
See all 19 Publications for this product

Customer reviews and Q&As

1-10 of 20 Abreviews or Q&A

Answer

Thank you for your note.

This is to let you know that I have just contacted and asked our Account Department to raise a credit note for you - for the cost of one vial of ab2768. For your information, the internal reference note for this credit is CN20942. You can use this credit in the near future for any of the products which are in the catalogue.

I hope this helps and if I can assist further, please do not hesitate to contact me.

Read More
Application
Immunocytochemistry/ Immunofluorescence
Blocking step
(agent) for 1 hour(s) and 0 minute(s) · Concentration: 3% · Temperature: 25°C
Sample
Rat Cell (Neuron)
Specification
Neuron
Permeabilization
Yes - 0.25% Triton-X
Fixative
Paraformaldehyde

Abcam user community

Verified customer

Submitted Jan 24 2014

Answer



We have a customer review (Abreview) using this antibody in IHC-Fr which notes successful use of enzymatic retrieval in rat brain sections.

Here is a link to this customer review:
https://www.abcam.com/Glucocorticoid-Receptor-BuGR2-antibody-ab2768/reviews/32407

Read More
Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Immunohistochemistry (PFA perfusion fixed frozen sections)
Sample
Rat Tissue sections (Brain)
Specification
Brain
Fixative
Formaldehyde
Antigen retrieval step
Enzymatic
Permeabilization
Yes - Triton X-100
Blocking step
BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 3% · Temperature: RT°C

Abcam user community

Verified customer

Submitted Oct 23 2012

Answer

I am glad you have found the information provide of use.

If you do have any further questions, please do not hesitate to contact us again.

Until then, I wish you all the best with your research.

Read More

Answer

Thank you for contacting us yesterday and sorry for the delay in getting back to you.

I can now share with you the information we have in regards to the epitope recognised by the Anti-Glucocorticoid Receptor antibody [BuGR2] (ab2768).

The epitope recognised by the antibody has been determined to be within the region 407 to 422 or Rat Glucocorticoid Receptor (SwissProt reference http://www.uniprot.org/uniprot/P06536). This corresponds to the region 388-402 of the human Glucocorticoid Receptor (SwissProt reference P04150). I would therefore expect the antibody to detect both the alpha and beta forms of the receptor. However, as we have not performed any studies to determine this conclusively I would not be able to guarantee this.

I hope this information has been of help. If you have any further questions, please do not hesitate to ask.

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Answer

Code de remise : ********
Date d’expiration : JJ MM AAAA
Je suis ravi que vous souhaitiez participer à notre offre, tester ab2768 en IHC-FoFr et partager vos résultats. Le code promotionnel correspondant est *******, il est valable 4 mois et pour la commande d’un autre anticorps primaire gratuit.
Vous devez dans un premier temps soumettre une Abreview avec vos résultats en IHC-FoFr en notant ce code dans la section « Additional notes ». Ce code sera ainsi activé et vous pourrez par la suite contacter notre service clientèle par téléphone pour passer votre prochaine commande, en vous munissant des informations suivantes : code de réduction, numéro de commande de ab2768.
Nous restons à votre disposition pour plus d’informations concernant cette offre promotionnelle. Dans l’attente de recevoir votre Abreview nous vous souhaitons bonne chance dans vos recherches.
Termes et Conditions : www.abcam.com/collaborationdiscount.

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Question
Answer

Merci de votre intérêt pour ab2768.
Comme expliqué par téléphone, cet anticorps a été testé en IHC-FoFr (PubMed: 20307510) mais nous ne disposons malheureusement pas d'image pour cette apllication.
Si vous souhaitez tester ab2768 en IHC-FoFr, il me sera possible de vous offrir une remise. Après soumission d’une Abreview avec vos résultats et une image, un anticorps primaire de votre choix vous sera offert.
Etapes à suivre :
1. Nous confirmer que vous souhaiteriez tester ab2768 en IHC-FoFr afin de recevoir le code de remise correspondant. Ce code doit être édité avant l’achat de ab2768
2. Acheter ab2768 par téléphone, fax ou internet (www.abcam.com)
3. Le tester en IHC-FoFr
4. Nous faire part de vos résultats grâce à notre système Abreview. Pour plus d’informations https://www.abcam.com/abreviews
5. Après soumission de votre Abreview, appeler notre service clientèle afin de passer votre prochaine commande grâce au code de réduction. Ce code de réduction est utilisable 120 jours après son édition et utilisable lors de l’achat d’un autre anticorps primaire.
N’hésitez pas à nous demander plus d’informations concernant cette offre promotionnelle.
Termes et Conditions : www.abcam.com/collaborationdiscount.

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Question

Dear technical support team:

This customer has purchased ab2768 (Anti-Glucocorticoid Receptor antibody [BuGR2]) and has conducted the wb with human sample sever times. The results show multiple bands, therefore, she wants to ask for your help to modify her experiment step, could you please offer any suggestion to help her?

I also attached her data in this letter and her experiment step as follow:



1. Order details:


Batch number: gr51696-3

Po: 1040660

Abcam product code: ab2768

Antibody storage conditions (temperature/reconstitution etc)


-20 ℃



2. Please describe the problem (high background, wrong band size, more bands, no band etc).

More bands

3. On what material are you testing the antibody in WB?

· Species: human

· What’s cell line or tissue: HK-2, human cell line

· Cell extract or Nuclear extract: both

· Purified protein or Recombinant protein: no



3. The lysate


How much protein was loaded: 60ug/ 30ug

What lysis buffer was used:



What protease inhibitors were used:

What loading buffer was used:

Phosphatase inhibitors

Did you heat the samples: temperature and time: 100℃ for 5 mins




4. Electrophoresis/Gel conditions/ Transfer conditions


Reducing or non reducing gel: Reducing gel

Reducing agent: SDS

Gel percentage : 7.5 %

Transfer conditions: (Type of membrane, Protein transfer verified):


PVDF, Protein transfer didn’t verified



5. Blocking conditions


Buffer: TTBS

Blocking agent: milk, BSA, serum, what percentage: 5% non-fat milk

Incubation time: at RT for 30 mins

Incubation temperature: at RT for 30 mins






6. Primary Antibody


Species: mouse

Reacts against: human


· At what dilution(s) have you tested this antibody: 1: 1000

· What dilution buffer was used: TTBS with 5% non-fat milk

· Incubation time: at 4℃ for overnight

· Incubation temperature: at 4℃ for overnight

· What washing steps were done:



7. Secondary Antibody


Species: goat

Reacts against: mouse

At what dilution(s) have you tested this antibody: 1:10000

Incubation time: at RT for 1 hr

Wash steps: at RT for 1 hr

Fluorochrome or enzyme conjugate: No

Do you know whether the problems you are experiencing come from the secondary? No, it isn’t come from the secondary.




8. Detection method
ECl, ECl+, other detection method:

ECL

9. Did you apply positive and negative controls along with the samples? Please specify.

To overexpress of GR

10. Optimization attempts

· How many times have you tried the Western? 3 times

· Have you eliminated the possibility that any background bands could be due to the secondary antibody? (Run a “No primary” control): No

· Do you obtain the same results every time e.g. are background bands always in the same place? Yes

· What steps have you altered?

1. GR can translocate to nucleus in DEX- treated.

2. To change protein conc.

3. To overexpress of GR.

Could you please help this customer to solve the problem?

Thanks for your kindly help

Best regards

Read More
Answer

Thank you for your enquiry regarding ab2768 and for taking the time to provide some useful details of the experiments. I am very sorry to hear that you are having problems with this antibody.

After reading through the detailed protocol you kindly forwarded to Abcam, I would like to make the following comments/suggestions:

1) Isoforms:

According to the literature, 9 different isoforms have been described :http://www.uniprot.org/uniprot/P04150 and it is possible that the customer may detect some splice variants.

2) Lysis buffer:

Could you please specify the compositions of the lysis buffer? Does it contain detergent?

3) Loading:

Has the customer tried to load less total protein than 30 ug per lane i.e. 5-10 ug.

4) Blocking:

Longer blocking than 30 mins would be recommended for this case such as 1 hour.

5) Positive control:

It would be worth running a positive control along with the samples such as liver or kidney tissue lysate (from mouse or human). As the on-line Western blot image indicates, total mouse liver lysate was used to test this antibody in the Lab.

I hope this will be useful for you. Should you still have any problem with this antibody after following these suggestions, then please do not hesitate to contact our Technical Department again.

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1-10 of 20 Abreviews or Q&A

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