The competition association binding method allows the characterization of the kinetics of unlabeled compounds and the calculation of receptor-drug affinity (K d). The K d value is defined as the ratio of the dissociation constant (or k off) of the receptor-bound ligand to its association rate constant (or k on) for a system at equilibrium. Traditionally, competition association binding experiments have been carried out using radiometric detection methods with limited assay throughput. Here we describe a novel method for the determination of unlabeled compound kinetics using the technique of time-resolved fluorescence resonance energy transfer (TR-FRET) performed at physiological temperature and sodium ion concentration. Based on a traditional screening format (10-point curves), up to 28 compounds can be tested on a single 384-well plate by this method.
Keywords: Association rate; Competition association binding kinetics; Dissociation rate; Fluorescent ligand; SNAP-tagged GPCR; Terbium cryptate; Time-resolved fluorescence resonance energy transfer.