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Analysis of Protein–Protein Interactions Using Bioluminescence Resonance Energy Transfer

  • Kevin D.G. Pfleger
Protocol
Part of the Methods in Molecular Biology™ book series (MIMB, volume 574)

Abstract

Knowledge of how and when proteins interact in living cells is fundamental to our understanding of cellular biology, and bioluminescence resonance energy transfer (BRET) provides an increasingly popular mechanism for studying these interactions in real time. The technique utilises heterologously expressed fusion proteins linking a bioluminescent donor or complementary acceptor fluorophore to proteins of interest. Resonance energy transfer between these fusion proteins is then detected when they are in close proximity, indicative of association either directly or as part of a complex. BRET is particularly useful for real-time monitoring of ligand-modulated interactions as dynamic changes in protein complex assembly can be observed in a live cell environment.

Key words

Bioluminescence resonance energy transfer, BRET protein–protein interaction, eBRET Renilla luciferase, Rluc8 fluorophore Venus 

Notes

Acknowledgements

The author would like to thank Professor Sanjiv Sam Gambhir and Dr. Atsushi Miyawaki for generously providing Rluc8 and Venus cDNA, respectively. The author and his work using the BRET methodology have been funded by the National Health and Medical Research Council of Australia in the form of a Peter Doherty Research Fellowship (#353709), Project Grants (#404087 and #566736) and a Development Grant (#513780).

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Copyright information

© Humana Press, a part of Springer Science+Business Media, LLC 2009

Authors and Affiliations

  • Kevin D.G. Pfleger
    • 1
  1. 1.Western Australian Institute for Medical Research (WAIMR) and Centre for Medical ResearchUniversity of Western AustraliaPerthAustralia

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