The TR-FRET ER alpha Coactivator Assay Kit provides a sensitive and robust method for high-throughput screening of potential estrogen receptor (ER) alpha ligands as agonists or antagonists of ligand-dependent coactivator recruitment. The kit uses an ER alpha ligand-binding domain (ER alpha-LBD) tagged with glutathione-S-transferase (GST), a terbium (Tb)-labeled anti-GST antibody, and a fluorescein-labeled coactivator peptide in a homogenous mix-and-read assay format.
Agonist Mode
To run the LanthaScreen TR-FRET ER alpha Coactivator Assay in agonist mode (to identify agonist compounds), ER alpha-LBD is added to ligand test compounds, followed by the addition of a mixture of fluorescein-coactivator peptide and Tb-anti-GST antibody. After incubation at room temperature, the TR-FRET 520: 495 nm emission ratio is calculated and used to determine the EC50 from a dose response curve of the compound. Based on the biology of the ER alpha-coactivator peptide interaction, this ligand EC50 is a composite value representing the amount of ligand required to bind to receptor, effect a conformational change, and recruit coactivator peptide.
Antagonist Mode
To run the LanthaScreen TR-FRET ER alpha Coactivator Assay in antagonist mode (to identify antagonist compounds), ER alpha-LBD is added to ligand test compounds, followed by addition of a mixture of agonist, fluorescein-coactivator peptide, and terbium (Tb)-labeled anti-GST antibody. The concentration of agonist used in this mode is the EC80 concentration as determined by first running the assay in agonist mode.