Human ER-alpha Transcription Factor Activity Assay

RayBio® Human ER-alpha Transcription Factor Activity Assay. This assay uses a dsDNA coated plate with canonical ER-alpha binding sequences to semi-quantitatively detect active ER-alpha in lysates or nuclear extracts. Dry ice shipment (additional fee).

$402.00  
TFEH-ERa-1
+

Antigen Information

Accession Number:
  • P03372
Gene ID:
  • 2099
Gene Symbols:
  • ESR
  • ESR1
  • NR3A1
Protein Name & Synonyms:
Estrogen receptor (ER), ER-alpha, Estradiol receptor, Nuclear receptor subfamily 3 group A member 1
Target Species:
  • Human

Assay Format

Specificity:
The olionucleotide/antibody pair provided in this kit recognizes mouse ER-alpha in whole lysates and nuclear extracts.
Number of Targets Detected:
1
Species Detected:
  • Human
Compatible Sample Types:
  • Cell Lysates
  • Nuclear Extracts
Design Principle:
  • Sandwich-based
Method of Detection:
Colorimetric
Quantitative/Semi-Quantitative:
Semi-Quantitative
Solid Support:
96-well Microplate

Product Specifications

Size:
1, 2, or 5 x 96-Well Strip Microplate Kit

Introduction

The estrogen receptor (ER) is a ligand-activated enhancer protein that belongs to the steroid/nuclear receptor superfamily and functions as a signal transducer and transcription factor to modulate expression of target genes. Mammalian ER is encoded by two genes: alpha and beta (ER-alpha and ER-beta) which have three major functional domains including an N-terminus that modulates transcription in a gene- and cell-specific manner through Activation Function-1, a highly conserved central DBD through which ER interacts directly with the DNA helix, and the LBD that contains Activation Function-2. In human physiology, ERs not only play a central role in the control of sexual behavior and reproductive functions, but are also widely involved in the differentiation of several tissues and organs, the modulation of inflammation, and brain and cardiovascular functions. In response to ligand binding of 17beta-estradiol (E2) or other agonists, ER changes conformation and dissociates from hsp90, hsp70 and other proteins, so that ligand-occupied ER dimer has been formed to regulate target genes. Activated ER stimulates target gene expression by two mechanisms: genomic and non-genomic (non-genotropic) pathways. In the genomic pathway, ligand-occupied ER directly binds to a specific sequence in the promoters of target genes (specifically, the estrogen response element or ERE), resulting in enhanced transcription. In the non-genomic pathway, ER can indirectly associate with promoters through protein-protein interactions with other DNA-binding transcription factors. For example, ER-alpha interacts with Sp1 in response to estrogen stimulation to regulate the transcription of RARalpha, insulin-like growth factor-binding protein-4, transforming growth factor alpha, LDL receptor genes, and others.

Product Features

  • Specific transcription factor-DNA binding assay
  • Perfect alternative to EMSA
  • Easy to perform in an ELISA format
  • Non-radioactive assay
  • High throughput (96-well plate format)
  • Assay can be completed within 5 hours

Application Notes

Kit Components
  • 96-well Strip Microplate pre-coated with DNA probes
  • DNA Binding Buffer
  • Positive Control Sample
  • Specific Competitor DNA probe
  • Non-specific Competitor DNA probe
  • Assay Reagent
  • DTT
  • Wash Buffer
  • Primary Antibody
  • HRP-conjugated Secondary Antibody
  • Antibody Diluent Buffer
  • TMB One-Step Substrate Reagent
  • Stop Solution
Other Materials Required
  • Distilled or deionized water
  • 100 ml and 1 liter graduated cylinders
  • Tubes to prepare sample dilutions
  • Absorbent paper
  • Precision pipettes to deliver 2 µl to 1 ml volumes
  • Adjustable 1-25 ml pipettes for reagent preparation
  • Microplate reader capable of measuring absorbance at 450 nm
Protocol Outline
  1. Prepare all reagents and samples as instructed in the manual.
  2. Add 100 µl of sample or positive control to each well.
  3. Incubate 2 h at RT or O/N at 4 °C.
  4. Add 100 µl of prepared primary antibody to each well.
  5. Incubate 1 h at RT.
  6. Add 100 µl of prepared HRP-secondary antibody to each well.
  7. Incubate 1 h at RT.
  8. Add 100 µl of TMB One-Step Substrate Reagent to each well.
  9. Incubate 30 min at RT.
  10. Add 50 µl of Stop Solution to each well.
  11. Read at 450 nm immediately.

Typical Data

Figure 1
Transcription factor assay of ER-alpha from nuclear extracts of MCF-7 cells or MCF-7 cells treated with H2O2 (200uM) for 3 hr with RayBio Activity Assay Kit.

Transcription factor assay of ER-alpha from nuclear extracts of MCF-7 cells or MCF-7 cells treated with H2O2 (200uM) for 3 hr with RayBio Activity Assay Kit

Figure 2
Transcription factor assay of ER-alpha from nuclear extracts of MCF-7 cells or MCF-7 cells treated with H2O2 (200uM) for 3 hr with the specific competitor or non-specific competitor. The result shows specific binding of ER-alpha to the conserved binding site detected by using RayBio ER-alpha TF Activity Assay Kit.

Transcription factor assay of ER-alpha from nuclear extracts of MCF-7 cells or MCF-7 cells treated with H2O2 (200uM) for 3 hr with the specific competitor or non-specific competitor. The result shows specific binding of ER-alpha to the conserved binding site detected by using RayBio ER-alpha TF Activity Assay Kit.

Storage/Stability

Upon receipt, the positive control should be removed and stored at -20° or -80°C. The remainder of the kit can be stored for up to 6 months at 2-8°C from the date of shipment. Opened Microplate Wells or reagents may be stored for up to 1 month at 2° to 8°C. Return unused wells to the pouch containing desiccant pack, reseal along entire edge.
Note: The kit can be used within one year if the whole kit is stored at -20°C upon receipt. Avoid repeated freeze-thaw cycles.

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Usage

This product is furnished for LABORATORY RESEARCH USE ONLY.
Not for diagnostic or therapeutic use.

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