Characterization of nuclear receptor ligands by multiplexed peptide interactions.

This unit describes a method to evaluate the effect that small molecules have on the binding interactions of a nuclear receptor protein with a series of peptides. The multiplexed microsphere-based system employs peptide-coupled microsphere populations that are fluorescently unique and thereby identifiable by flow cytometric analysis. Up to 100 different peptide-nuclear receptor interactions may be analyzed in a single well of a 96-well microtiter plate. This approach allows rapid and sensitive characterization of nuclear receptor ligands based on nuclear receptor protein-peptide interaction profiles. Since nuclear receptor binding interactions are dynamically related to protein conformation, the approach allows rapid evaluation of nuclear receptor ligands that may impart unique protein structure. The no-wash format and the high surface density of the microsphere-coupled interaction partner offer a moderately high-throughput system to examine low- to high-affinity interactions with excellent sensitivity. This approach, although described for nuclear receptors, may also be applied to other types of molecular interactions.