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Monitoring 14-3-3 Protein Interactions with a Homogeneous Fluorescence Polarization Assay

Department of Pharmacology, Emory University School of Medicine and Emory Chemistry-Biology Discovery Center, Emory University, Atlanta, GA

Shane C. Masters

Department of Pharmacology, Emory University School of Medicine and Emory Chemistry-Biology Discovery Center, Emory University, Atlanta, GA; Medical College of Georgia, Augusta, GA

Fadlo R. Khuri

Winship Cancer Institute, Emory University School of Medicine, Atlanta, GA

Haian Fu

Department of Pharmacology, Emory University School of Medicine and Emory Chemistry-Biology Discovery Center, Emory University, Atlanta, GA 30322; Winship Cancer Institute, Emory University School of Medicine, Atlanta, GA hfu{at}emory.edu

The 14-3-3 proteins mediate phosphorylation-dependent protein-protein interactions. Through binding to numerous client proteins, 14-3-3 controls a wide range of physiological processes and has been implicated in a variety of diseases, including cancer and neurodegenerative disorders. To better understand the structure and function of 14-3-3 proteins and to develop small-molecule modulators of 14-3-3 proteins for physiological studies and potential therapeutic interventions, the authors have designed and optimized a highly sensitive fluorescence polarization (FP)–based 14-3-3 assay. Using the interaction of 14-3-3 with a fluorescently labeled phosphopeptide from Raf-1 as amodel system, they have achieved a simple 1-step "mixand-measure" method for analyzing 14-3-3 proteins. This is a solution-based, versatile method that can be used tomonitor the binding of 14-3-3with a variety of client proteins. The 14-3-3 FP assay is highly stable and has achieved a robust performance in a 384-well format with a demonstrated signal-to-noise ratio greater than 10 and a Z• factor greater than 0.7. Because of its simplicity and high sensitivity, this assay is generally applicable to studying 14-3-3/client-protein interactions and especially valuable for high-throughput screening of 14-3-3 modulators.

Key Words: 14-3-3 • fluorescence polarization • protein-protein interaction

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