Analysis of Protein–Protein Interactions Using Bioluminescence Resonance Energy Transfer

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


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 



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).


  1. 1.
    Pfleger, K. D. G., and Eidne, K. A. (2006) Illuminating insights into protein-protein interactions using bioluminescence resonance energy transfer (BRET). Nat Methods 3, 165–174.PubMedCrossRefGoogle Scholar
  2. 2.
    Pfleger, K. D. G., Seeber, R. M., and Eidne, K. A. (2006) Bioluminescence resonance energy transfer (BRET) for the real-time detection of protein–protein interactions. Nat Protoc 1 , 337–345.CrossRefGoogle Scholar
  3. 3.
    Milligan, G., and Bouvier, M. (2005) Methods to monitor the quaternary structure of G-protein-coupled receptors. FEBS J 272, 2914–2925.PubMedCrossRefGoogle Scholar
  4. 4.
    Wu, P., and Brand, L. (1994) Resonance energy transfer: methods and applications. Anal Biochem 218 , 1–13.PubMedCrossRefGoogle Scholar
  5. 5.
    Pfleger, K. D. G., and Eidne, K. A. (2003) New technologies: bioluminescence resonance energy transfer (BRET) for the detection of real time interactions involving G-protein coupled receptors. Pituitary 6 , 141–151.PubMedCrossRefGoogle Scholar
  6. 6.
    Pfleger, K. D. G., and Eidne, K. A. (2005) Monitoring the formation of dynamic G protein-coupled receptor–protein complexes in living cells. Biochem J 385 , 625–637.PubMedCrossRefGoogle Scholar
  7. 7.
    De, A., Loening, A. M., and Gambhir, S. S. (2007) An improved bioluminescence resonance energy transfer strategy for imaging intracellular events in single cells and living subjects. Cancer Res 67 , 7175–7183.PubMedCrossRefGoogle Scholar
  8. 8.
    Nagai, T., Ibata, K., Park, E. S., Kubota, M., Mikoshiba, K., and Miyawaki, A. (2002) A variant of yellow fluorescent protein with fast and efficient maturation for cell-biological applications. Nat Biotechnol 20, 87–90.PubMedCrossRefGoogle Scholar
  9. 9.
    Pfleger, K. D. G., Dromey, J. R., Dalrymple, M. B., Lim, E. M. L., Thomas, W. G., and Eidne, K. A. (2006) Extended bioluminescence resonance energy transfer (eBRET) for monitoring prolonged protein–protein interactions in live cells. Cell Signal 18 , 1664–1670.PubMedCrossRefGoogle Scholar
  10. 10.
    Kocan, M., See, H.B., Seeber, R. M., Eidne, K. A., and Pfleger, K. D. G. (2008) Demonstration of improvements to the bioluminescence resonance energy transfer (BRET) technology for the mointoring of G protein-coupled receptors in live cells. J Biomol Screen 13, 888–898.PubMedGoogle Scholar
  11. 11.
    Hamdan, F. F., Audet, M., Garneau, P., Pelletier, J., and Bouvier, M. (2005) High-throughput screening of G protein-coupled receptor antagonists using a bioluminescence resonance energy transfer 1-based beta-arrestin2 recruitment assay. J Biomol Screen 10 , 463–475.PubMedCrossRefGoogle Scholar
  12. 12.
    Mercier, J. F., Salahpour, A., Angers, S., Breit, A., and Bouvier, M. (2002) Quantitative assessment of β1- and β2-adrenergic receptor homo- and heterodimerization by bioluminescence resonance energy transfer. J Biol Chem 277 , 44925–44931.PubMedCrossRefGoogle Scholar
  13. 13.
    Germain-Desprez, D., Bazinet, M., Bouvier, M., and Aubry, M. (2003) Oligomerization of transcriptional intermediary factor 1 regulators and interaction with ZNF74 nuclear matrix protein revealed by bioluminescence resonance energy transfer in living cells. J Biol Chem 278 , 22367–22373.PubMedCrossRefGoogle Scholar
  14. 14.
    Breit, A., Lagace, M., and Bouvier, M. (2004) Hetero-oligomerization between β2- and β3-adrenergic receptors generates a β-adrenergic signalling unit with distinct functional properties. J Biol Chem 279, 28756–28765.PubMedCrossRefGoogle Scholar
  15. 15.
    Kroeger, K. M., Hanyaloglu, A. C., Seeber, R. M., Miles, L. E., and Eidne, K. A. (2001) Constitutive and agonist-dependent homo-oligomerization of the thyrotropin-releasing hormone receptor. Detection in living cells using bioluminescence resonance energy transfer. J Biol Chem 276 , 12736–12743.PubMedCrossRefGoogle Scholar
  16. 16.
    Ayoub, M. A., Couturier, C., Lucas-Meunier, E., Angers, S., Fossier, P., Bouvier, M., and Jockers, R. (2002) Monitoring of ligand-independent dimerization and ligand-induced conformational changes of melatonin receptors in living cells by bioluminescence resonance energy transfer. J Biol Chem 277 , 21522–21528.PubMedCrossRefGoogle Scholar
  17. 17.
    Zhang, J. -H., Chung, T. D. Y., and Oldenburg, K. R. (1999) A simple statistical parameter for use in evaluation and validation of high throughput screening assays. J Bio Mol Screen 4 , 67–73.CrossRefGoogle Scholar
  18. 18.
    Angers, S., Salahpour, A., Joly, E., Hilairet, S., Chelsky, D., Dennis, M., and Bouvier, M. (2000) Detection of β2-adrenergic receptor dimerization in living cells using bioluminescence resonance energy transfer (BRET). Proc Natl Acad Sci USA 97, 3684–3689.Google Scholar
  19. 19.
    McVey, M., Ramsay, D., Kellett, E., Rees, S., Wilson, S., Pope, A. J., and Milligan, G. (2001) Monitoring receptor oligomerization using time-resolved fluorescence resonance energy transfer and bioluminescence resonance energy transfer. J Biol Chem 276, 14092–14099.Google Scholar
  20. 20.
    James, J. R., Oliveira, M. I., Carmo, A. M., Iaboni, A., and Davis, S. J. (2006) A rigorous experimental framework for detecting protein oligomerization using bioluminescence resonance energy transfer. Nat Methods 3 , 1001–1006.PubMedCrossRefGoogle Scholar
  21. 21.
    Bouvier, M., Heveker, N., Jockers, R., Marullo, S., and Milligan, G. (2007) BRET analysis of GPCR oligomerization: newer does not mean better. Nat Methods 4 , 3–4.PubMedCrossRefGoogle Scholar
  22. 22.
    Salahpour, A., and Masri, B. (2007) Experimental challenge to a ‘rigorous’ BRET analysis of GPCR oligomerization. Nat Methods 4 , 599–600.PubMedCrossRefGoogle Scholar

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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