This kit contains Goat Tb-Anti-GST antibody; other kit components are the same as kit A15125:
The LanthaScreen™ 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 a terbium (Tb)-labeled anti-GST antibody, a fluorescein-labeled coactivator peptide, and an ER alpha ligand-binding domain (ER alpha-LBD) that is tagged with glutathione-S-transferase (GST), in a homogenous mix-and-read assay format.
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 addition of a mixture of the fluorescein-coactivator peptide and Tb-anti-GST antibody. After an incubation period at room temperature, the TR-FRET 520:495 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 (Figure 1).
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 (Figure 2).
Contents and Storage:
The LanthaScreen™ TR-FRET ER alpha Coactivator Assay Kit contains the ER alpha-LBD (GST) protein, fluorescently labeled PGC1a coactivator peptide, Tb-anti-GST antibody, and buffers. Store components as indicated in the assay protocol (-80°C, -20°C, or +4°C).