Overview

  • Product name
    Estrogen 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

    Estrogen Receptor Transcription Factor Assay Kit (Colorimetric) (ab207203) is a high throughput assay to quantify Estrogen Receptor (ER) 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 Estrogen Receptor consensus binding site (5’ –GGTCACAGTGACC– 3’) has been immobilized onto a 96-well plate. Active Estrogen Receptor present in the nuclear extract specifically binds to the oligonucleotide. Estrogen Receptor is detected by a primary antibody that recognizes an epitope of Estrogen 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 Estrogen 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 – 20 µg nuclear extract/well.

  • Notes

    ER belongs to the superfamily of ligand-inducible transcription factors. Human ERa is comprised of 595 amino acids and displays an approximate molecular weight of 66-70 kDa. Six functional regions have been identified. A hypervariable domain (aa 1-184) contains activation function 1 (AF1). The DNA binding domain (DBD, aa 185-263) contains two zinc finger motifs and is highly conserved across the nuclear receptor superfamily. It is responsible for the binding of the receptor to estrogen response elements (EREs) and contributes to dimerization and activation. Typically, EREs consist of two inverted half-sites separated by 3 bp (5´-GGTCAnnnTGACC-3´). The region which separates the ligand binding domain (LBD) and the DBD is called the hinge region (aa 264-302).

    The LBD (aa 303-553) consists of 12 a-helices, which form a hydrophobic pocket responsible for ligand binding. The function of the final domain (aa 554-595) is not clear but is thought to play a role in distinguishing between agonist and antagonist binding. Human ERb is expressed as multiple isoforms. Structure and function studies have shown that the DBD of ERa and ERb are highly homologous, approaching 96%, whereas the LBD showed only 59% homology. The general mechanism of action of ERb is thought to be similar to that of ERa. ERa and ERb have the ability to interact with target promoters in three different complexes: ERa homodimers, ERb homodimers and ERa/ERb heterodimers.

    The transcriptional effects of ER can be mediated through several mechanisms other than E2-ER complexes binding to EREs. E2-ER complexes can also trans activate genes through protein-protein interactions with transcription factors such as AP-1 or Sp1 that bind DNA, with coaccessory proteins (Src, ACTR), some of which have histone acetylase activity, and with RNA Polymerase II complex proteins. In addition, ERs serve to repress genes, which also plays an important role in E2 action.

  • Platform
    Microplate reader

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 ER 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
    ERa antibody 1 x 11µl 1 x 55µl
    Lysis Buffer 1 x 10ml 1 x 50ml
    MCF-7 nuclear extract (2.5 µg/µL) 1 x 40µl 1 x 200µl
    Mutated oligonucleotide (10 pmol/µL) 1 x 100µl 1 x 500µl
    Plate sealer 1 unit 5 units
    Poly [d(l-c)] (17 µg/μL) 1 x 100µl 1 x 500µl
    Protease Inhibitor Cocktail 1 x 100µl 1 x 500µl
    Stop Solution 1 x 11ml 1 x 55ml
    Wild-type oligonucleotide (10 pmol/µL) 1 x 100µl 1 x 500µl
  • Research areas
  • Function
    Nuclear hormone receptor. The steroid hormones and their receptors are involved in the regulation of eukaryotic gene expression and affect cellular proliferation and differentiation in target tissues. Can activate the transcriptional activity of TFF1.
  • 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
    Phosphorylated by cyclin A/CDK2. Phosphorylation probably enhances transcriptional activity.
    Glycosylated; contains N-acetylglucosamine, probably O-linked.
    Ubiquitinated. Deubiquitinated by OTUB1.
    Dimethylated by PRMT1 at Arg-260. The methylation may favor cytoplasmic localization.
    Palmitoylated (isoform 3). Not biotinylated (isoform 3).
  • Cellular localization
    Nucleus. Cytoplasm. Cell membrane. A minor fraction is associated with the inner membrane and Nucleus. Cytoplasm. Cell membrane. Associated with the inner membrane via palmitoylation.
  • Information by UniProt
  • Alternative names
    • Atherosclerosis, susceptibility to, included
    • DKFZp686N23123
    • ER
    • ER Alpha
    • ER Beta
    • ER-alpha
    • ER[a]
    • ER[b]
    • Era
    • ERalpha
    • Erb
    • Erb2
    • ERbeta
    • ESR
    • ESR BETA
    • ESR1
    • ESR1_HUMAN
    • ESR2
    • ESRA
    • ESRB
    • Estr
    • Estra
    • Estradiol receptor
    • Estradiol Receptor alpha
    • Estradiol Receptor beta
    • ESTRB
    • Estrogen receptor
    • Estrogen Receptor 1
    • Estrogen receptor 1 (alpha)
    • Estrogen Receptor 2
    • Estrogen receptor 2 (ER beta)
    • Estrogen receptor 2 ER beta
    • Estrogen receptor alpha
    • Estrogen receptor beta 4
    • Estrogen resistance, included
    • HDL cholesterol, augmented response of, to hormone replacement, included
    • Myocardial infarction, susceptibility to, included
    • NR3A1
    • NR3A2
    • Nuclear receptor subfamily 3 group A member 1
    • Nuclear receptor subfamily 3 group A member 2
    • OTTHUMP00000017718
    • OTTHUMP00000017719
    • RNESTROR
    see all
  • Database links

Images

  • Different amounts of untreated (grey) and hydrogen peroxide treated (black) MCF-7 were tested for ERα activity. These results are provided for demonstration purposes only.

Protocols

References

ab207203 has not yet been referenced specifically in any publications.

Customer reviews and Q&As

There are currently no Customer reviews or Questions for ab207203.
Please use the links above to contact us or submit feedback about this product.

Please note: All products are "FOR RESEARCH USE ONLY AND ARE NOT INTENDED FOR DIAGNOSTIC OR THERAPEUTIC USE"
For licensing inquiries, please contact partnerships@abcam.com

Sign up