Abstract
Bioluminescence resonance energy transfer (BRET) has become a widely used technique to monitor protein–protein interactions. It involves resonance energy transfer between a bioluminescent donor and a fluorescent acceptor. Because the donor emits photons intrinsically, fluorescence excitation is unnecessary. Therefore, BRET avoids some of the problems inherent in fluorescence resonance energy transfer (FRET) approaches, such as photobleaching, autofluorescence, and undesirable stimulation of photobiological processes. In the past, BRET signals have generally been too dim to be effectively imaged. Newly available cameras that are more sensitive coupled to image splitter now enable BRET imaging in plant and mammalian cells and tissues. In addition, new substrates and enhanced luciferases enable brighter signals that allow even subcellular BRET imaging. Here, we report methods for BRET imaging of (1) localization of COP1 dimerization in plant cells and tissues and (2) subcellular distributions of interactions of the CCAAT/Enhancer Binding Protein α (C/EBPα) in single mammalian cells. We also discuss methods for the correction of BRET images for tissues that absorb light of different spectra. This progress should catalyze further applications of BRET for imaging and high-throughput assays.
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Acknowledgments
We thank Dr. R.N. Day for mouse GHFT1 cells and C/EBP244 fused to EYFP, Dr. Roger Tsien for Venus YFP, Stein Servick for technical assistance, Drs. Yao Xu, Michael Geusz, David Piston & Shin Yamazaki for advice concerning BRET techniques, imaging techniques, and image analysis, and Drs. Keith Wood & Erika Hawkins of Promega for making ViviRenTM and PBI 1419 available to us prior to its commercial release. This work was supported by NSF grant MCB-0114653 to Drs. Albrecht von Arnim and Carl Johnson as part of the Arabidopsis 2010 project, and the following grants to Dr. Carl Johnson: NSF SGER grant # IOS-0854942, National Institutes of General Medical Science R01 GM065467, and National Institute of Mental Health R21 MH 080035.
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Xie*, Q., Soutto*, M., Xu*, X., Zhang, Y., Johnson, C.H. (2011). Bioluminescence Resonance Energy Transfer (BRET) Imaging in Plant Seedlings and Mammalian Cells. In: Shah, K. (eds) Molecular Imaging. Methods in Molecular Biology, vol 680. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-60761-901-7_1
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DOI: https://doi.org/10.1007/978-1-60761-901-7_1
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