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Nucleic Acids Research 2005 33(6):e61; doi:10.1093/nar/gni057
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Published online 1 April 2005

© The Author 2005. Published by Oxford University Press. All rights reserved
The online version of this article has been published under an open access model. Users are entitled to use, reproduce, disseminate, or display the open access version of this article for non-commercial purposes provided that: the original authorship is properly and fully attributed; the Journal and Oxford University Press are attributed as the original place of publication with the correct citation details given; if an article is subsequently reproduced or disseminated not in its entirety but only in part or as a derivative work this must be clearly indicated. For commercial re-use, please contact journals.permissions{at}oupjournals.org

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Determination of tumor necrosis factor receptor-associated factor trimerization in living cells by CFP->YFP->mRFP FRET detected by flow cytometry

Liusheng He*, Xiaoli Wu, James Simone, Derek Hewgill and Peter E. Lipsky1

Flow Cytometry Section, Office of Science and Technology, National Institutes of Health Bethesda, MD 20892, USA 1Autoimmunity Branch, National Institute of Arthritis and Musculoskeletal and Skin Diseases, National Institutes of Health Bethesda, MD 20892, USA

*To whom correspondence should be addressed. Tel: +1 301 594 3531; Fax: +1 301 402 2209; Email: Lihe{at}mail.nih.gov

Received February 7, 2005. Revised March 8, 2005. Accepted March 8, 2005.

The availability of protein fluorophores with appropriate spectral properties has made it possible to employ fluorescence resonance energy transfer (FRET) to assess interactions between three proteins microscopically. Flow cytometry offers excellent sensitivity, effective signal separation and the capacity to assess a large number of events, and, therefore, should be an ideal means to explore protein interactions in living cells. Here, we report a flow-cytometric FRET technique that employed both direct energy transfer from CFP->YFP->mRFP and donor quenching to assess TRAF2 trimerization in living cells. Initially, a series of fusion proteins incorporating CFP, YFP and mRFP with spacers that did or did not permit FRET were employed to document the magnitude of CFP->YFP and YFP->mRFP FRET and to calculate the efficiency of CFP->YFP->mRFP two-step FRET. Based upon this, TRAF2 homotrimerization could be detected. This method should have great utility in studying the dynamics of interactions between three specific proteins in vivo.


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