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Epigenetics and Nuclear Signaling Transcription Domain Families Zinc Finger
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Estrogen Receptor Transcription Factor Assay Kit (Colorimetric) (ab207203)

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  • Protocol Booklet
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Different amounts of untreated (Gray) and H2O2 (Black) post-treated nuclear extracts from MCF-7 cells are tested for ERa activity.

    Key features and details

    • Assay type: Semi-quantitative
    • Detection method: Colorimetric
    • Platform: Microplate reader
    • Assay time: 3 hr 30 min
    • Sample type: Nuclear Extracts
    • Sensitivity: 600 ng/well

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    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

      • Epigenetics and Nuclear Signaling
      • Transcription
      • Domain Families
      • Zinc Finger
      • Signal Transduction
      • Signaling Pathway
      • Nuclear Signaling
      • Nuclear Hormone Receptors
      • Estrogen
      • Neuroscience
      • Endocrine system
      • Gonadotrophic axis
      • Epigenetics and Nuclear Signaling
      • Nuclear Signaling Pathways
      • Nuclear Receptors
      • Estrogen
      • Cancer
      • Signal transduction
      • Nuclear signaling
      • Nuclear hormone receptors
      • Estrogen
      • Cancer
      • Tumor biomarkers
      • Receptors
    • 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.
    • Target information above from: UniProt accession P03372 The UniProt Consortium
      The Universal Protein Resource (UniProt) in 2010
      Nucleic Acids Res. 38:D142-D148 (2010) .

      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

      • Entrez Gene: 2099 Human
      • Entrez Gene: 2100 Human
      • Entrez Gene: 13982 Mouse
      • Entrez Gene: 24890 Rat
      • Omim: 601663 Human
      • Omim: 133430 Human
      • SwissProt: P03372 Human
      • SwissProt: Q92731 Human
      • SwissProt: P19785 Mouse
      • SwissProt: P06211 Rat
      • Unigene: 208124 Human
      • Unigene: 463262 Mouse
      • Unigene: 9213 Mouse
      • Unigene: 10595 Rat
      see all

    Associated products

      Images

      • Different amounts of untreated (Gray) and H2O2 (Black) post-treated nuclear extracts from MCF-7 cells are tested for ERa activity.
        Different amounts of untreated (Gray) and H2O2 (Black) post-treated nuclear extracts from MCF-7 cells are tested for ERa activity.

        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

      • Protocol Booklet

      Click here to view the general protocols

      Datasheets and documents

      • Datasheet
      • SDS
    • References (0)

      Publishing research using ab207203? Please let us know so that we can cite the reference in this datasheet.

      ab207203 has not yet been referenced specifically in any publications.

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