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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:
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.
|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|
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.
|Functional Studies||Use at an assay dependent concentration.|
ab207207 has not yet been referenced specifically in any publications.
Please note: All products are "FOR RESEARCH USE ONLY AND ARE NOT INTENDED FOR DIAGNOSTIC OR THERAPEUTIC USE"