Abstract
Fluorescence polarization (FP) technology is based on the measurement of molecule rotation, and has been widely used to study molecular interactions in solution. This method can be used to measure binding and dissociation between two molecules if one of the binding molecules is relatively small and fluorescent. The fluorescently labeled small molecule (such as a small peptide) rotates rapidly in the solution. Upon excitation by polarized light, the emitted light remains depolarized and gives rise to a low FP signal. When the fluorescent small molecules in solution are bound to bigger molecules (such as a protein), the movement of the complex becomes slower. When such a complex is excited with polarized light, much of the emitted light is polarized because of the slow movement of the complex. Thus, the binding of a fluorescently labeled small molecule to a bigger molecule can be monitored by the change in polarization and measured by the generation of an increased FP signal. This chapter aims to provide a step-by-step practical procedure for developing an FP assay in a multi-well plate format to monitor protein-protein interaction (PPI) in a homogenous format.
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Acknowledgments
I thank Dr. Haian Fu for his insightful comments and members of the Fu laboratory for constructive suggestions. This work was supported in part by grants from the National Institutes of Health/National Institute of General Medical Sciences (R01 GM53165 to H.F.) and a Lung Cancer program seed grant from the Winship Cancer Institute. Yuhong Du is a recipient of the Emory Drug Development and Pharmacogenomics Academy Research Fellowship.
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Du, Y. (2015). Fluorescence Polarization Assay to Quantify Protein-Protein Interactions in an HTS Format. In: Meyerkord, C., Fu, H. (eds) Protein-Protein Interactions. Methods in Molecular Biology, vol 1278. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-2425-7_35
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DOI: https://doi.org/10.1007/978-1-4939-2425-7_35
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-2424-0
Online ISBN: 978-1-4939-2425-7
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