Estrogen Receptor Alpha (ERα; NR3A1)
|Product Family||Product Number||Product Description||Technical Manual|
|IB00401-32||Human ERα Reporter Assay System, 3 x 32 assays in 96-well format||Technical Manual|
|IB00401||Human ERα Reporter Assay System, 1 x 96-well format assays||Technical Manual|
|IB00402||Human ERα Reporter Assay System, 1 x 384-well format assays||Technical Manual|
Estrogen Receptor Alpha Assay Kit
This Estrogen Receptor Alpha (ERα) assay kit is an all-inclusive ER alpha reporter assay system that includes, in addition to ER Alpha Reporter Cells, two optimized media for use during cell culture and (optionally) in diluting the test samples, a reference agonist, Luciferase Detection Reagent, a cell culture-ready assay plate, and a detailed protocol.
ER Alpha Reporter Cells are prepared using INDIGO’s proprietary CryoMite™ process. This cryo-preservation method yields high cell viability post-thaw, and provides the convenience of immediately dispensing healthy, division-competent reporter cells into assay plates. There is no need for intermediate spin-and-wash steps, viability determinations, or cell titer adjustments.
INDIGO’s ERα assay kits feature a luciferase detection reagent specially formulated to provide stable light emission between 5 and 90+ minutes after initiating the luciferase reaction. Incorporating a 5-minute reaction-rest period ensures that light emission profiles attain maximal stability, thereby allowing assay plates to be processed in batch. By doing so, the signal output from all sample wells, from one plate to the next, may be directly compared within an experimental set.
Kits are offered in different assay formats to accommodate researchers’ needs: 3 x 32, 1 x 96, and 1 x 384 assay formats for screening small numbers of test compounds, as well as custom bulk reagents for HTS applications.
Bulk assay reagents can be custom manufactured to accommodate any scale of HTS. Please inquire.
ERa assay kits also available in: Zebrafish
Estrogen Receptor Alpha Assay Services
The primary application of INDIGO’s cell-based nuclear receptor assays are to quantitatively assess the bioactivity of a test compound as an agonist (activator) or antagonist (inhibition of an agonist response) of a given receptor. Service assays include a positive control reference compound and ‘vehicle’ control for every experiment. A formal study report and all data files are provided to the client upon completion of the study. To receive a quote for your proposed study, complete & submit the online “Request a Quote” form or contact an INDIGO Customer Service Representative to discuss your desired study parameters.
Service Assays: Human, Zebrafish
Estrogen Receptor Alpha Background
ER-alpha is encoded by the human gene ESR1 (Estrogen Receptor 1). The estrogen receptor (ESR) is a ligand-activated transcription factor composed of several domains important for hormone binding, DNA binding, and activation of transcription. Alternative splicing results in several ESR1 mRNA transcripts, which differ primarily in their 5-prime untranslated regions. The translated receptors show less variability.
INDIGO's Estrogen Receptor Alpha Reporter Assay Systems utilize proprietary mammalian cells engineered to express constitutive, high-level expression of the full length Human Estrogen Receptor 1 (NR3A1), a ligand-dependent transcription factor commonly referred to as ERα.
INDIGO's Estrogen Receptor Alpha Reporter Cells include the luciferase reporter gene functionally linked to an ERα-responsive promoter. Thus, quantifying changes in luciferase expression in the treated reporter cells provides a sensitive surrogate measure of the changes in ERα activity. Luciferase gene expression occurs after ligand-bound ERα undergoes nuclear translocation, DNA binding, recruitment, and assembly of the co-activators and accessory factors required to form a functional transcription complex, culminating in expression of the target gene. Unlike some other cell-based assay strategies, the readout from INDIGO's reporter cells demands the same orchestration of all intracellular molecular interactions and events that can be expected to occur in vivo.
The principle application of this assay product is in the screening of test samples to quantify functional activities, either agonist or antagonist, that they may exert against the human estrogen receptor alpha.
For more information on ERα, visit the Nuclear Receptor Resource.
Synonyms: Estrogen Receptor Alpha, ERα, ER alpha, ESR1, Estrogen Receptor 1, ERa, ESRA, ER-alpha, NR3A1
Estrogen Receptor Alpha Assay Data
Analyses of Human ERα Reporter Cells using 17-β-Estradiol (provided), PPT (4,4'4"-(4-Propyl-[1H]-pyrazole-1,3,5-triyl tris-phenol; Tocris), and (R,R)-THC ([R,R]-5,11-diethyl-5,6,11,12-tetrahydro-2,8-chrysenediol; Tocris). Concentrated stocks of agonists were prepared in DMSO; the highest concentration of DMSO in assay wells was 0.1%. Luminescence was quantified and values of average relative light units (RLU) and corresponding standard deviation (SD) were determined for each treatment concentration (n ≥ 3). Fold-activation and Z’ values were calculated as described by Zhang, et al. (1999). Non-linear regression curve-fitting of the data and EC50 determinations were performed using GraphPad Prism software.
Human ERα antagonist assays were performed using Endoxifen, Tamoxifen citrate, ICI182780 and MPP dihydrochloride (all from Tocris). INDIGO’s Live Cell Multiplex Assay (#LCM-01) was also performed to confirm that the observed drop in RLU values resulted from receptor-specific inhibition, and not from compound-induced cell death (data not shown). Reporter cells were co-treated with a fixed concentration (3.2 nM, approximating EC75) of the agonist 17β-Estradiol and varying concentrations of respective challenge antagonists.