Abstract
Bioluminescent Resonance Energy Transfer is a naturally occurring phenomenon that can be exploited to explore protein−protein interactions in real-time in intact cells and cellular extracts. It detects energy transferred between a bioluminescent donor enzyme (Renilla luciferase) fusion protein and a fluorescent (GFP2, a mutant of Green Fluorescent Protein) acceptor fusion protein. Optimal detection of BRET2 energy transfer relies on the distance and orientation generated by the fusion proteins. This chapter describes in detail the BRET2 assay as it is used to examine the physical interaction between the nuclear receptor ERα and the transcriptional coactivator SRC-1. Description of methods include selection of donor and acceptor combinations, fusion construct generation and validation, cell culture and transfection, individual fluorescence and luminescence detection, BRET2 detection, and data analysis.
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Acknowledgments
Financial Support: This project was supported by R01DK068432 and a grant from the Louisiana Cancer Research Consortium to BGR, and by an individual NRSA 1F31CA126489 and a Graduate Alliance for Education in Louisiana (GAELA) Fellowship to TTD.
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© 2009 Humana Press, a part of Springer Science+Business Media, LLC
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Duplessis, T.T., Koterba, K.L., Rowan, B.G. (2009). Detection of ERα-SRC-1 Interactions Using Bioluminescent Resonance Energy Transfer. In: Park-Sarge, OK., Curry, T. (eds) Molecular Endocrinology. Methods in Molecular Biology, vol 590. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-60327-378-7_16
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DOI: https://doi.org/10.1007/978-1-60327-378-7_16
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Publisher Name: Humana Press, Totowa, NJ
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