Time-resolved fluorescence resonance energy transfer

To determine the binding affinity of XN and TXN to PPARγ, a Lanthascreen TR-FRET PPARγ competitive binding assay was performed by Thermo Fisher Scientific (cite manual) (Lanthascreen, Invitrogen). A terbium-labeled anti-GST antibody binds to a GST-PPARγ-ligand-binding domain fusion protein in which the LBD is occupied by a fluorescent pan-PPAR ligand (Fluormone Pan-PPAR Green). Energy transfer from the antibody to the ligand occurs and a high TR-FRET ratio (emission signal at 520 nm/495 nm) is detected. When a test compound displaces the ligand from PPARγ-LBD, a decrease in the FRET signal occurs and a lower TR-FRET ratio is detected (Corporation, 2008). For each compound (XN, TXN, or oleic acid), a 10-point serial dilution (250,000–12.5 nM) was tested. Binding curves were generated by plotting percent displacement versus log concentration (nM), and IC₅₀ values were determined using a sigmoidal dose response (variable slope).