This kit contains Goat Tb-Anti-GST antibody; other kit components are the same as kit A15132:
The LanthaScreen™ TR-FRET GR (glucocorticoid receptor) Coactivator Assay provides a sensitive and robust method for high-throughput screening of potential GR 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 a human recombinant GR ligand-binding domain (GR-LBD) that is tagged with glutathione-S-transferase (GST) in a homogeneous mix-and-read assay format.
Agonist mode: When running the LanthaScreen™ TR-FRET GR Coactivator Assay in agonist mode (to identify agonist compounds), GR-LBD is added to ligand test compounds followed by addition of a mixture of the fluorescein-coactivator peptide and Tb-labeled 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 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 1).
Antagonist mode: When the LanthaScreen™ TR-FRET GR Coactivator Assay is run in antagonist mode (to identify antagonist compounds), GR-LBD is added to ligand test compounds followed by addition of a mixture of agonist, fluorescein-coactivator peptide, and Tb-labeled anti-GST antibody (Figure 2). The concentration of agonist used in this mode is the EC80 concentration as determined by first running the assay in agonist mode.
Contents and Storage: The LanthaScreen™ TR-FRET GR Coactivator Assay Kit contains GR-LBD (GST) protein, fluorescently labeled SRC1-4 coactivator peptide, Tb-labeled anti-GST antibody, and buffers. Store components as indicated in the assay protocol (-80°C, -20°C, or 4°C).
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