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Bioluminescence resonance energy transfer (BRET) for the real-time detection of protein-protein interactions

Nature Protocols volume 1, pages 337–345 (2006)Cite this article

Abstract

A substantial range of protein-protein interactions can be readily monitored in real time using bioluminescence resonance energy transfer (BRET). The procedure involves heterologous coexpression of fusion proteins, which link proteins of interest to a bioluminescent donor enzyme or acceptor fluorophore. Energy transfer between these proteins is then detected. This protocol encompasses BRET1, BRET2 and the recently described eBRET, including selection of the donor, acceptor and substrate combination, fusion construct generation and validation, cell culture, fluorescence and luminescence detection, BRET detection and data analysis. The protocol is particularly suited to studying protein-protein interactions in live cells (adherent or in suspension), but cell extracts and purified proteins can also be used. Furthermore, although the procedure is illustrated with references to mammalian cell culture conditions, this protocol can be readily used for bacterial or plant studies. Once fusion proteins are generated and validated, the procedure typically takes 48–72 h depending on cell culture requirements.

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Figure 1: An illustration of the BRET protocol using a microplate luminometer.
Figure 2: Examples of theoretical BRET data from ligand (reagent) modulated interactions.

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Authors and Affiliations

  1. 7TM Laboratory/Laboratory for Molecular Endocrinology, Western Australian Institute for Medical Research (WAIMR) and UWA Centre for Medical Research, University of Western Australia, Nedlands, Perth, 6009, Western Australia, Australia

    Kevin D G Pfleger, Ruth M Seeber & Karin A Eidne

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  1. Kevin D G Pfleger
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Correspondence to Kevin D G Pfleger.

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Pfleger, K., Seeber, R. & Eidne, K. Bioluminescence resonance energy transfer (BRET) for the real-time detection of protein-protein interactions. Nat Protoc 1, 337–345 (2006). https://doi.org/10.1038/nprot.2006.52

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