|Description:||The TR-FRET Constitutive Androstane Receptor (CAR) Coactivator Assay provides a sensitive and robust method for highthroughput screening (HTS) of potential CAR ligands as agonists of ligand-dependent coactivator recruitment or inverse agonists of liganddependent coactivator displacement. The kit uses a terbium-labeled anti-GST antibody, a fluorescein-labeled coactivator peptide, and a CAR ligand-binding domain (CAR-LBD) that is tagged with glutathione-S-transferase (GST) in a homogeneous mix-and-read assay format.
When running the TR-FRET CAR Coactivator Assay, CAR-LBD is added to ligand test compounds followed by addition of a mixture of the fluorescein-coactivator peptide and terbium anti-GST antibody. After an incubation period at room temperature, the 520 nm/ 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 CAR-coactivator peptide interaction, this ligand’s EC50 is a composite value representing the amount of ligand required to bind to receptor, effect a conformational change, and either recruit or displace coactivator peptide.
|Applications:||Co-Factor Interaction Assay|
|Storage:||The TR-FRET CAR Coactivator Assay Kit contains CAR-LBD (GST) protein, fluorescently labeled PGC1a coactivator peptide, terbium-labeled anti-GST antibody, and buffers. Store components as indicated in the assay protocol (-80°C, -20°C, or +4°C).|
|Size:||800 x 20 µL assays|
|Materials Required but Not Supplied:||Microplate Reader|