Fluorescence Polarization (FP) Assays for Monitoring Peptide-Protein or Nucleic Acid-Protein Binding

The technique of fluorescence polarization (FP) is based on the observation that when a fluorescently labeled molecule is excited by polarized light, it emits light with a degree of polarization that is inversely proportional to the rate of molecular rotation. This property of fluorescence can be used to measure the interaction of a small labeled ligand with a larger protein and provides a basis for direct and competition binding assays. FP assays are readily adaptable to a high-throughput format, have been used successfully in screens directed against a wide range of targets, and are particularly valuable in screening for inhibitors of protein-protein and protein-nucleic acid interactions when a small binding epitope can be identified for one of the partners. The protocols in this article describe a general procedure for development of FP assays to monitor binding of such a peptide or oligonucleotide to a protein of interest. Curr. Protoc. Chem Biol. 1:1-15. © 2009 by John Wiley & Sons, Inc.