NR Peptide ERα ELISA

Applications

The NR Peptide ERα ELISA has many applications including:

• Quantitative study of compounds affecting ERα binding (agonist or antagonist)
• Analysis of compounds exhibiting tissue-selective ERα agonist/antagonist properties
• Analysis of ligand combinations and their effect on ERα activation

Features

• Results in about 3 hours
• Quantitative, non-radioactive detection
• Specific, antibody-based measurement
• Works with cell or tissue extracts and recombinant proteins
• High throughput compatible
• Optimized Capture Peptide for high affinity capture of ligand-activated ER

Evaluation of nuclear receptor agonist/antagonist compounds

The quantitative results provided by the NR Peptide ELISAs make it easy to accurately evaluate the effects of various agonist or antagonist compounds on NR activation. To demonstrate, the NR Peptide ERα ELISA Kit was used to measure estrogen receptor alpha activation in response to several well-characterized agonist and antagonist compounds (Figure 1).

Figure 1: ERα agonism/antagonism dose-response curve of diethylstilbestrol (DES), estradiol, tamoxifen and clomiphene.

Nuclear extracts from the breast cancer cell line MCF-7 are incubated in the presence of the test compounds (100-fold serial dilutions: from 10^-6 to 10^-14 M) in wells of the NR Peptide ERα ELISA plate. Only ligand-activated ER can bind to the Capture Peptide. This bound ER is specifically detected with ERα antibody. Secondary antibody and detection solution are then used to quantitate ligand-activated ER.

Nuclear receptors (NR) belong to a superfamily of structurally related ligand-inducible transcription factors whose members include receptors for estrogen (ER), glucocorticoid (GR), androgen (AR), thyroid hormone (TR), progesterone (PR) and vitamin D (VDR). Inappropriate signaling by these receptors is associated with numerous disease states, including cancer, asthma, infertility, osteoporosis and arthritis.

Why use the Nuclear Receptor ELISAs?

• NR Peptide measures ligand-activated NR
• Quantitative results in just hours
• ELISA format eliminates the need to run, blot and develop gels
• Analyze multiple samples in low volume, high throughput
• Ability to assay both cellular extracts and recombinant proteins

The NR Peptide ELISA method

Upon ligand-receptor binding, nuclear receptors undergo a conformational change, which can result in recruitment of co-activators (agonists) or co-repressors (antagonists). Depending on the nuclear receptor and the tissue location, a ligand may have both agonist and antagonist properties, resulting in different biological outcomes.

Active Motif's NR Peptide ELISAs are specifically designed to capture ligand-activated nuclear receptor. Each NR Peptide Kit provides a 96-well plate coated with Capture Peptide that includes the consensus-binding motif of the nuclear receptor's co-activator. Addition of sample results in binding of activated nuclear receptor to the immobilized Capture Peptide. Each well is then incubated with a specific primary antibody. Subsequent incubation with HRP-conjugated antibody and developing solution provides an easily quantified readout.

Figure 1: Quantitative measurement of ligand-activated nuclear receptor.

Ligand-activated NR contained in the sample binds to the Capture Peptide immobilized in the well. Addition of primary and secondary antibodies and detection solution provides quantitative measurement of the bound, ligand-activated nuclear receptor.

Contents & Storage

One or five 96-well plate(s) coated with optimized nuclear receptor Capture Peptide, ERα primary antibody, HRP-conjugated secondary antibody, positive control cell extract, Binding, Wash and Antibody Dilution Buffers and Detection Solutions. Reagent storage conditions vary from room temperature to -80°C, see manual for details. All reagents are guaranteed stable for 6 months.