The LanthaScreen® TR-FRET Glucocorticoid Receptor Coactivator Assay Kit provides a sensitive and robust method for high-throughput screening of potential glucocorticoid receptor (GR) ligands as agonists or antagonists of ligand-dependent coactivator recruitment. The kit uses a human recombinant GR ligand-binding domain (GR-LBD) tagged with glutathione-S-transferase (GST) (also available separately), a terbium (Tb)-labeled anti-GST antibody, and a fluorescein-labeled coactivator peptide in a homogeneous mix-and-read assay format.
When running the LanthaScreen TR-FRET Glucocorticoid Receptor Coactivator Assay in agonist mode (to identify agonist compounds), GR-LBD is added to ligand test compounds, followed by the addition of a mixture of fluorescein-coactivator peptide and Tb-labeled anti-GST antibody. After incubation at room temperature, the 520/495 nm TR-FRET ratio is calculated and can be used to determine the EC50 from a dose response curve of the compound. Based on the biology of the GR-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 (see figure).
When the LanthaScreen TR-FRET Glucocorticoid Receptor Coactivator Assay is run in antagonist mode (to identify antagonist compounds), GR-LBD is added to ligand test compounds followed by the addition of a mixture of agonist, fluorescein-coactivator peptide, and Tb-labeled anti-GST antibody (see figure). The concentration of agonist used in this mode is the EC80 concentration as determined by first running the assay in agonist mode.