Assay Method Information

Assay Name:  Scintillation Proximity Assay (IC50) and PPAR gamma Transactivation Assay (EC50)
Description:  Human 6His-PPAR ligand-binding domain was added to the mixture containing radioligand and test compound, followed by yttrium silicate polylysine SPA beads (Amersham). The plates were sealed with press-on adhesive sealer and covered with aluminum foil. The plates were incubated at room temperature while shaking at 700 rpm on an IKA-Schuttler MST 4 titer plate shaker for one hour. After 30 min of settling, radioligand displacement was measured using a Wallac scintillation counter (Wallac Trilux 1450 Microbeta Liquid Scintillation and Luminescence counter). Ligand binding was calculated as percent displacement of total radioligand binding (DMSO control). The binding signal (cpm) in duplicates was plotted as a function of compound concentrations (M) and the plot was fitted to an equation by non-linear regression and IC50 derived from those plots using Spotfire. All experiments were repeated twice and the geometric mean was calculated for each IC50. HepG2 cells were transfected under standard conditions with Gal4RE-luciferase, Gal4:PPAR LBD and CMV beta-gal using Fugene 6. Transfected cells were treated with compounds for 18-24 h, lysed and luciferase and beta-gal assays performed (Promega) using a Dynex luminometer and Molecular Devices Plate reader. Luciferase values were corrected for transfection efficiency using beta-gal. Normalized luciferase values were plotted against dose and EC50 values were determined using GraphPad Prism.
Affinity data for this assay

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