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Illuminating insights into protein-protein interactions using bioluminescence resonance energy transfer (BRET)

Abstract

Bioluminescence resonance energy transfer (BRET) is a straightforward biophysical technique for studying protein-protein interactions. It requires: (1) that proteins of interest and suitable controls be labeled with either a donor or acceptor molecule, (2) placement of these labeled proteins in the desired environment for assessing their potential interaction, and (3) use of suitable detection instrumentation to monitor resultant energy transfer. There are now several possible applications, combinations of donor and acceptor molecules, potential assay environments and detection system perturbations. Therefore, this review aims to demystify and clarify the important aspects of the BRET methodology that should be considered when using this technique.

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Figure 1
Figure 2: An illustration of a typical BRET protocol, with energy transfer detected in live cells by a microplate reader.
Figure 3: Schematic representations illustrating the overlap of normalized Rluc emission spectra with normalized GFP excitation and emission spectra, together with typical filter combinations.
Figure 4: Qualitative BRET competition data for a constitutive interaction.
Figure 5: Examples of BRET saturation curves.

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Acknowledgements

We acknowledge U. Schmidt for providing spectral data, M. Dalrymple, R. Seeber and G. Pfleger for proofreading the manuscript, and L. Coleman for assistance with manuscript preparation. The authors' work using the BRET methodology is funded by the National Health and Medical Research Council (NHMRC) of Australia (project grants #254646, #303256 and #404087). K.D.G.P. and K.A.E. are supported by NHMRC Peter Doherty (#353709) and Principal Research (#212064) Fellowships, respectively.

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Correspondence to Kevin D G Pfleger.

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K.A.E. was involved with the development of the Mithras instrument (Berthold Technologies) for BRET detection. K.D.G.P. and K.A.E. have been involved in the testing of the EnduRen substrate (Promega) for use with BRET and, in the past, Promega have contributed to the conference expenses of K.D.G.P. No payments in terms of salaries have been received and neither author receives any commission for the sale of these products.

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Pfleger, K., Eidne, K. Illuminating insights into protein-protein interactions using bioluminescence resonance energy transfer (BRET). Nat Methods 3, 165–174 (2006). https://doi.org/10.1038/nmeth841

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