Glucocorticoid Receptor Transcription Factor Assay Kit (Colorimetric) (ab207207)

Overview

  • Product name
    Glucocorticoid Receptor Transcription Factor Assay Kit (Colorimetric)
  • Detection method
    Colorimetric
  • Sample type
    Nuclear Extracts
  • Assay type
    Semi-quantitative
  • Sensitivity
    < 600 ng/well
  • Assay time
    3h 30m
  • Species reactivity
    Reacts with: Mouse, Rat, Human
  • Product overview

    Glucocorticoid Receptor Transcription Factor Assay Kit (Colorimetric) (ab207207) is a high throughput assay to quantify Glucocorticoid Receptor (GR) activation in nuclear extracts. This assay combines a quick ELISA format with a sensitive and specific non-radioactive assay for transcription factor activation.


    A specific double stranded DNA sequence containing the Glucocorticoid Receptor consensus binding site (5’ –GGTACAnnnTGTTCT– 3’) has been immobilized onto a 96-well plate. Active Glucocorticoid Receptor present in the nuclear extract specifically binds to the oligonucleotide. Glucocorticoid Receptor is detected by a primary antibody that recognizes an epitope of Glucocorticoid Receptor accessible only when the protein is activated and bound to its target DNA. An HRP-conjugated secondary antibody provides sensitive colorimetric readout that at OD 450 nm. This product detects human, mouse and rat Glucocorticoid Receptor.


    Key performance and benefits:



    • Assay time: 3.5 hours (cell extracts preparation not included).

    • Detection limit: < 0.6 µg nuclear extract/well.

    • Detection range: 0.6 – 10 µg nuclear extract/well.

  • Notes

    Glucocorticoids can affect a large number of metabolic, cardiovascular, immune, inflammatory and behavioral functions. They are produced by the adrenal cortex and are under the control of the hypothalamus and pituitary (hypothalamus-pituitary-adrenal [HPA] axis). At the cellular level, glucocorticoid effects are mediated by the Glucocorticoid Receptor (GR). GR belongs to the superfamily of nuclear hormone receptors that includes receptors for estrogens, progestins, vitamin D and thyroid hormone.

    The nuclear hormone receptors share a characteristic three-domain structure. The N-terminal activates target genes and interacts with transcription machinery. Two highly conserved zinc fingers constitute the DNA-binding domain and also participate in dimerization, nuclear translocation and transactivation. The C-terminal contains the ligand-binding domain, and also includes sequences important for heat shock protein (hsp) binding, nuclear translocation, dimerization and transactivation.  

    The unliganded GR is part of a multiprotein complex that consists of the receptor, two molecules of hsp90 and one molecule each of hsp70 and hsp56. Glucocorticoids, when present, are able to cross the cell membrane and interact with GR. When bound, there is a conformational change in the GR molecule that results in dissociation from the hsp complex, hyper-phosphorylation of GR and unmasking of nuclear localization signals. When in the nucleus, the activated GR can act in two ways: directly with specific DNA sequences or indirectly with other transcription factors. GR mutations can result in glucocorticoid resistance or hypersensitivity, and can cause severe disturbances in mood, pathologic alterations of metabolism and, correspondingly, hypotension or hypertension and excessive or suppressed inflammatory/immune responses.

  • Tested applications
    Suitable for: Functional Studiesmore details

Properties

  • Storage instructions
    Please refer to protocols.
  • Components 1 x 96 tests 5 x 96 tests
    10X Antibody Binding Buffer 1 x 2.2ml 1 x 11ml
    10X Wash Buffer 1 x 22ml 1 x 110ml
    96-well GR assay plate 1 unit 5 units
    Anti-rabbit HRP-conjugated IgG (0.25 μg/μL) 1 x 11µl 1 x 55µl
    Binding Buffer 1 x 10ml 1 x 50ml
    Developing Solution 1 x 11ml 1 x 55ml
    Dithiothreitol (DTT) (1 M) 1 x 100µl 1 x 500µl
    GR antibody 1 x 11µl 1 x 55µl
    HeLa nuclear extract (2.5 μg/μL) 1 x 40µl 1 x 200µl
    Herring sperm DNA (1 μg/μL) 1 x 100µl 1 x 500µl
    Lysis Buffer 1 x 10ml 1 x 50ml
    Mutated oligonucleotide(20 pmol/μL) 1 x 100µl 1 x 500µl
    Plate sealer 1 unit 5 units
    Protease Inhibitor Cocktail 1 x 100µl 1 x 500µl
    Stop Solution 1 x 11ml 1 x 55ml
    Wild-type oligonucleotide (20 pmol/μL) 1 x 100µl 1 x 500µl
  • Research areas
  • 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
  • Alternative names
    • GCCR
    • GCR
    • GCR_HUMAN
    • GCRST
    • glucocorticoid nuclear receptor variant 1
    • Glucocorticoid receptor
    • GR
    • GRL
    • Grl1
    • nr3c1
    • Nuclear receptor subfamily 3 group C member 1
    • nuclear receptor subfamily 3, group C, member 1 (glucocorticoid receptor)
    see all
  • Database links

Applications

Our Abpromise guarantee covers the use of ab207207 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
Functional Studies Use at an assay dependent concentration.

Images

  • Different amounts of nuclear extracts from untreated (grey) and Dexamethasone-treated (black) HeLa cells were tested for GR activity. These results are provided for demonstration purposes only.

Protocols

References

ab207207 has not yet been referenced specifically in any publications.

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Please note: All products are "FOR RESEARCH USE ONLY AND ARE NOT INTENDED FOR DIAGNOSTIC OR THERAPEUTIC USE"

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