Overview

Properties

Applications

Our Abpromise guarantee covers the use of ab3581 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 Use a concentration of 5 µg/ml.

Fixation: 4% paraformaldehyde for 15 minutes at room temperature. Permeabilisation: PBS containing 0.01% saponin for 15 mins Blocking: 1% BSA in PBS Primary: overnight at 4ºC

EMSA Use at an assay dependent concentration.
IHC-P Use at an assay dependent concentration. PubMed: 20438742Sample Preparation: endogenous peroxidase blocked with 0.3% hydrogen peroxide. Antigen retrieval: Heat induced antigen retrieval performed in 0.01M Tris-EDTA pH 9.0. Primary antibody: overnight at 4°C
Flow Cyt Use at an assay dependent concentration.

ab171870 - Rabbit polyclonal IgG, is suitable for use as an isotype control with this antibody.

 

ICC 1/1500.
IP Use at an assay dependent concentration. Sample preparation: RIPA buffer containing protease inhibitors was added to cell lysate. Primary antibody: 1:10 dilution for 2 h on ice Prewashed magnetic beads added to lysate mixture and incubated overnight at +4ºC. After washing, sample reducing buffer was added and boiled (5 min, 95ºC)
WB 1/500. Detects a band of approximately 90 kDa (predicted molecular weight: 83 kDa).Can be blocked with Human Glucocorticoid Receptor beta peptide (ab39765). Sample preparation: Lysates boiled in reducing buffer for 5 min. 20µg of protein loaded Blocking agent: 5% non-fat milk in Tris buffered saline (TBS) Primary antibody: overnight at 4°C

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
    • glucocorticoid nuclear receptor variant 1 antibody
    • Glucocorticoid receptor antibody
    • Glucocorticoid receptor beta isoform antibody
    • GR antibody
    • GRL antibody
    • nr3c1 antibody
    • Nuclear receptor subfamily 3 group C member 1 antibody
    see all

Images

  • ICC/IF image of ab3581 stained HeLa cells. The cells were 4% formaldehyde fixed (10 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 (ab3581, 5µg/ml) overnight at +4°C. The secondary antibody (green) was Alexa Fluor® 488 goat anti-rabbit 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.

  • All lanes : Anti-Glucocorticoid Receptor beta antibody (ab3581) at 1/500 dilution

    Lane 1 : COS-74 whole cell extract transfected with Glucocorticoid Receptor alpha
    Lane 2 : COS-74 whole cell extract transfected with Glucocorticoid Receptor beta

    Predicted band size: 83 kDa

References

This product has been referenced in:
  • Butler CA  et al. Glucocorticoid receptor ß and histone deacetylase 1 and 2 expression in the airways of severe asthma. Thorax 67:392-8 (2012). ICC/IF ; Human . Read more (PubMed: 22156779) »
  • Melarangi T  et al. Glucocorticoid resistance in chronic lymphocytic leukaemia is associated with a failure of upregulated Bim/Bcl-2 complexes to activate Bax and Bak. Cell Death Dis 3:e372 (2012). WB ; Human . Read more (PubMed: 22898870) »
See all 5 Publications for this product

Customer reviews and Q&As

1-5 of 5 Abreviews or Q&A

Question
Answer

Thank you for contacting us.

In addition to sending a free of charge replacement of ab3581, I have discussed this issue further with the lab. When using this new antibody we have a few extra suggestions which may help with your results.

1. Try heating the samples at 70 degrees for 20 minutes rather than boiling for 10 minutes.

2. You could also try switching your blocking buffer to 5% BSA.

I hope that these suggestions will improve your results.

We would also be very grateful if you could submit an Abreview about this product via the online product datasheet once you have retested it. We always encourage customers to send their results back to us, whether positive or negative, and we make all product information available to researchers.

To find out more about our Abreview system, please see the following webpage:

https://www.abcam.com/abreviews

We hope to receive your Abreview shortly. Thank you for your time and effort.

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Answer

Thank you for confirming these details and for your cooperation. The details provided enable us to closely monitor the quality of our products.

I am sorry this product did not perform as stated on the datasheet and for the inconvenience this has caused. As requested, I have issued a free of charge replacement with the order number XXXXX.

To check the status of the order please contact our Customer Service team and reference this number.

Please note that this free of charge replacement vial is also covered by our Abpromise guarantee. Should you still be experiencing difficulties, or if you have any further questions, please do not hesitate to let us know.

I wish you the best of luck with your research.

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Answer

Thank you for taking time to complete our questionnaire and for contacting us. I am sorry to hear this antibody is not providing satisfactory results.

Unfortunately Mass Spectrometry was not performed on the extra bands to know weather these are really degradation products or truncated versions of the GR beta protein.

Having reviewed the protocol details, I have no suggestions to make as this is exactly how I would have performed the Western blot. I would like to thank you for bringing this antibody to our attention and thanks to the details provided, we are now in the process of reviewing this product to see whether we would like to keep it in our catalogue or replace it.

I appreciate the time you have spent in the laboratory and I apologize for the inconvenience caused.

I am pleased to offer you a free of charge replacement, credit note, or refund in compensation. If you would like a free of charge replacement, may I suggest ab130227 (https://www.abcam.com/ab130227)?

Thank you for your cooperation. I look forward to hearing from you with details of how you would like to proceed.

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Question

Dear Sir or Madam,

I would have a couple of technical questions regarding one of your product, Glucocorticoid-Receptor-beta-antibody-ab3581, ( link : https://www.abcam.com/Glucocorticoid-Receptor-beta-antibody-ab3581.html )

I use / used your Glucocorticoid-Receptor-beta-antibody for western blot .

1. On the web page the sample photo you show there are two bands- why is that if the antibody is specific for Glucocorticoid-Receptor-beta only.
Which one is the band for GR beta ?
Did you get any other bands , which seemed to specific band below these two bands?

2. I use Caco2 cell line which was transfected with human Glucocorticoid-Receptor-beta but I always get extra bands at around 50, little over 25 kd and if the exposure time last little longer even around 37 KD both in the normal Caco2 and the transfected one, which are stronger than the band we believe the GR beta band should be. We also experienced very strong bands in normal HeLa cell line at 50, little over 25 kd .

Do you have have any experience with the cell lines-especially Caco-2- and your Glucocorticoid-Receptor-beta-antibody-ab3581 and did you also have detected the above mentioned extra bands? Could you give us any possible explanation why do we get these results repetadly even though we handle our samples with special care and try to prevent protein degradation as much as we possible.

The seconder antibody which we use is goat anti-rabbit IgG-HRP: SC-2004. Link : http://www.scbt.com/datasheet-2004-goat-anti-rabbit-igg-hrp.html

We would be really grateful if you could give us some assistance and possible answers regarding our questions.

Read More
Answer

Thank you for contacting us.

I have contacted the lab who have provided me with the original western blot which I have attached to this email.

As can be seen on the image, we do see some faint bands of smaller molecular weight. We are not certain as to the identity of these bands, but it has been reported in the literature that GR beta is often shown as a doublet and therefore, these bands are possibly representing a degradation product or truncated version of the protein.

The antibody is specific to Glucocorticoid-Receptor-beta as its immunogen is amino acids xxxxxxxxx (xxxxxxxxxx) which shows only a 26.6% sequence similarity with Glucocorticoid-Receptor-alpha. We believe that the band of interest is the upper band on the blot but we are unsure as to the identity of the lower band.

Unfortunately, we do not have any experience using cell lines Caco-2- with the Glucocorticoid-Receptor-beta-antibody so I can not give you any more explanation as to what these extra bands you are seeing are. However, if you fill out the questionnaire I sent you previously and return it to me, I will be more than happy to have a look through it and suggest possible improvements.

If the product was purchased in the last six months and is being used according to our Abpromise, we would be happy to replace or refund the antibody.

I hope this information is helpful to you. Please do not hesitate to contact us if you need any more advice or information.

Use our products? Submit an Abreview. Earn rewards!
https://www.abcam.com/abreviews

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Question

Dear Sir or Madam,

I would have a couple of technical questions regarding one of your product, Glucocorticoid-Receptor-beta-antibody-ab3581, ( link : https://www.abcam.com/Glucocorticoid-Receptor-beta-antibody-ab3581.html )

I use / used your Glucocorticoid-Receptor-beta-antibody for western blot .

1. On the web page the sample photo you show there are two bands- why is that if the antibody is specific for Glucocorticoid-Receptor-beta only.
Which one is the band for GR beta ?
Did you get any other bands , which seemed to specific band below these two bands?

2. I use Caco2 cell line which was transfected with human Glucocorticoid-Receptor-beta but I always get extra bands at around 50, little over 25 kd and if the exposure time last little longer even around 37 KD both in the normal Caco2 and the transfected one, which are stronger than the band we believe the GR beta band should be. We also experienced very strong bands in normal HeLa cell line at 50, little over 25 kd .

Do you have have any experience with the cell lines-especially Caco-2- and your Glucocorticoid-Receptor-beta-antibody-ab3581 and did you also have detected the above mentioned extra bands? Could you give us any possible explanation why do we get these results repetadly even though we handle our samples with special care and try to prevent protein degradation as much as we possible.

The seconder antibody which we use is goat anti-rabbit IgG-HRP: SC-2004. Link : http://www.scbt.com/datasheet-2004-goat-anti-rabbit-igg-hrp.html

We would be really grateful if you could give us some assistance and possible answers regarding our questions.

Read More
Answer

Thank you for contacting us.

I am sorry to hear you are experiencing difficulties with one of our products. We take product complaints very seriously, and investigate every product that we feel may not be performing correctly. I have contacted the lab to see whether they have any explications as to what these extra bands on the western blot might be.

In the meantime, I am attaching our questionnaire so that we can gather further information regarding the samples tested and the protocol used. Once we receive the completed questionnaire, we will look at the protocol and see if there are any suggestions we can make that may improve the results. This information will also allow us to investigate this case internally and initiate additional testing where necessary. If the product was purchased in the last six months and is being used according to our Abpromise, we would be happy to replace or refund the antibody.

I look forward to receiving your reply.

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