This kit contains Goat Tb-Anti-GST antibody; other kit components are the same as kit A15119:
The LanthaScreen™ TR-FRET Androgen Receptor (AR) Coactivator Assay Kit provides a sensitive and robust method for high-throughput screening of potential 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 terbium (Tb)-labeled anti-GST antibody, a fluorescein-labeled coactivator peptide, and a rat AR ligand-binding domain (AR-LBD) that is tagged with hexahistidine and glutathione-S-transferase (GST).
How it works: Agonist mode
When the LanthaScreen™ TR-FRET AR Coactivator Assay is run in agonist mode (to identify agonist compounds), AR-LBD is added to ligand test compounds, followed by addition of a mixture of the fluorescein-coactivator peptide and Tb-anti-GST antibody. After room temperature incubation, the TR-FRET 520:495 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 (Figure 1).
How it works: Antagonist mode
When the LanthaScreen™ TR-FRET AR 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. An example of data produced in the antagonist mode is shown in Figure 2.
Contents and Storage:
The LanthaScreen™ TR-FRET AR Coactivator Assay Kit contains AR-LBD (His and GST) protein, fluorescein-labeled D11 FxxLF coactivator peptide, Tb-anti-GST antibody, and buffers. Store components as indicated in the assay protocol (-80°C, -20°C, or +4°C).