| Assay Method Information | |
| | Binding of Examples to Transthyretin - EC50 Determination |
| Description: | TTR SPA binding assays were performed in a final volume of 60 mI containing 100 ng human TTR (biotinylated recombinant protein) coupled to 25 pg SPA beads (streptavidin coated, Perkin Elmer, RPNQ0007) and 50 nM [3H] tafamidis (Moravek, MT-1003033), plus varying concentrations of test compound or vehicle. Briefly, assays were prepared at room temperature in 384-well plates (Corning, 3767) containing 200 nl_ of test compound in DMSO (or DMSO as vehicle). The plates also contained wells with a saturating concentration of unlabeled ligand (200 nl_ of 3 mM tafamidis or 3 mM thyroxine in DMSO) for measuring non-specific binding. Assays were initiated by addition of 20 mI of 5 mg/mL TTR protein in assay buffer (10 mM Tris pH 7.5, 150 mM NaCI, 0.25% Triton X-100) and 20 mI_ of 150 nM [3H] tafamidis in assay buffer. The plates were incubated 1 hour prior to addition of 20 mI_ of 1.25 mg/ml_ SPA beads diluted in assay buffer. The assays were incubated an additional 10 hours to allow binding to reach equilibrium and the amount of receptor-ligand complex was determined by liquid scintillation counting using a 1450 Microbeta Trilux (Wallac). The % effect values for test wells were calculated based on the total binding (vehicle, 0% effect) and non-specific binding (unlabeled ligand, 100% effect) wells on each assay plate. ECso values were then determined using a standard 4 parameter logistic dose response equation. |
| Affinity data for this assay | |
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