|Description:||The TR-FRET Androgen Receptor Coactivator Assay Kit provides a sensitive and robust method for high-throughput screening of potential androgen receptor (AR) ligands as agonists of coactivator recruitment or antagonists of agonist-dependent coactivator recruitment. In both the agonist and antagonist modes, the homogeneous mix-and-read assay uses a rat AR ligand-binding domain (AR-LBD) tagged with hexahistidine and glutathione-S-transferase (GST) (also available separately), a terbium (Tb)-labeled anti-GST antibody, and a fluorescein-labeled coactivator peptide.
When the LanthaScreen TR-FRET Androgen Receptor Coactivator Assay is run in agonist mode (to identify agonist compounds), AR-LBD is added to ligand test compounds, followed by the addition of a mixture of fluorescein-coactivator peptide and Tb-anti-GST antibody. After room temperature incubation, the TR-FRET 520: 495 nm emission ratio is calculated and used to determine the EC50 from a dose response curve of the compound. 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.
When the LanthaScreen TR-FRET Androgen Receptor Coactivator Assay is run in antagonist mode (to identify antagonist compounds), AR-LBD is added to ligand test compounds, followed by addition of a mixture of DHT agonist, fluorescein-coactivator peptide, and Tb-anti-GST antibody. The concentration of DHT agonist used in this mode is the EC80 concentration as determined by first running the assay in agonist mode.
|Applications:||Co-Factor Interaction Assay, Nuclear Receptor Assay|
|Storage:||1 tube AR Ligand Binding Domain Recombinant Protein (store at -68 to -85°C)
1 tube Peptide (store at -5 to -30°C)
2 bottles TR-FRET Buffer (store at -5 to -30°C)
1 tube Tb-Anti-GST Ab (store at -5 to -30°C)
1 tube of DTT (store at -5 to -30°C)
|Size:||800 x 20 µL assays|