This kit contains Goat Tb-Anti-GST antibody; other kit components are the same as kit A15131:
The LanthaScreen™ TR-FRET PR (Progesterone Receptor) Coactivator Assay provides a sensitive and robust method for high-throughput screening of potential PR 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 PR ligand--binding domain (PR-LBD) that is tagged with glutathione-S-transferase (GST), in a homogenous mix-and-read assay format.
When using the LanthaScreen™ TR-FRET PR Coactivator Assay in agonist mode (to identify agonist compounds), PR-LBD is added to ligand test compounds followed by addition of a mixture of the fluorescein-labeled coactivator peptide and Tb-labeled anti-GST antibody. After an incubation period at room temperature, the TR-FRET ratio of 520/495 is calculated and can be used to determine the EC50 from a dose response curve of the compound. Based on the biology of the PR-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).
When using the LanthaScreen™ TR-FRET PR Coactivator Assay in antagonist mode (to identify antagonist compounds), PR-LBD is added to ligand test compounds followed by addition of a mixture of agonist, fluorescein-labeled coactivator peptide, and terbium-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: LanthaScreen™ TR-FRET PR Coactivator Assay kits contain PR-LBD (GST) protein, fluorescently labeled SRC1-4 coactivator peptide, Tb-anti-GST antibody, and buffers. Store components as indicated in the assay protocol (–80°C, –20°C or 4°C).
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