Simultaneous Monitoring of Discrete Binding Events Using Dual-Acceptor Terbium-Based LRET

Abstract

Lanthanide resonance energy transfer (LRET) between chelated terbium and two spectrally distinct acceptor fluorophores (fluorescein and Alexa Fluor 633) is demonstrated. Ligand-dependent association and dissociation of differentially labeled peptides derived from coactivator and corepressor proteins with PPAR isoforms was monitored simultaneously, with no mathematical deconvolution required to separate the signals from the distinct acceptor fluorophores. These results demonstrate the utility of terbium chelates for the analysis of complex multicomponent binding events by LRET.