Monitoring 14-3-3 protein interactions with a homogeneous fluorescence polarization assay.
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 a model system, they have achieved a simple 1-step "mix-and-measure" method for analyzing 14-3-3 proteins. This is a solution-based, versatile method that can be used to monitor the binding of 14-3-3 with 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.
Du
Yuhong
Y
Department of Pharmacology, Emory University School of Medicine and Emory Chemistry-Biology Discovery Center, Emory University, Atlanta, GA 30322, USA.
Masters
Shane C
SC
Khuri
Fadlo R
FR
Fu
Haian
H
D048948:14-3-3 Proteins / Q000378:metabolism*; D016231:Fluorescence Polarization Immunoassay; D011485:Protein Binding
D048948:14-3-3 Proteins