2.19. Time-resolved fluorescence resonance energy transfer (TR-FRET) assays in vitro

To measure ligand binding to PPARα, TR-FRET was performed with a LanthaScreen TR-FRET PPARα competitive binding assay according to the manufacturer's instructions (Invitrogen). Briefly, a serial dilution of AM6545 and GW7647, a selective PPARα agonist, was prepared in a 384-well polypropylene assay plate. Fluormone Pan-PPAR Green, PPARα-LBD, and terbium (Tb)-labeled anti-GST Abs were then added to each sample. The assay plate was incubated for 2 h at room temperature prior to measuring the 520 nm/495 nm emission ratio of each well using an Infinite F500 microplate reader (Tecan, Männedorf, Switzerland) with instrument settings as described in the manufacturer's instructions for LanthaScreen assays.