Measuring Förster Resonance Energy Transfer
or biosensor probe activities. T is non- invasive imaging technique provides a method to verify the results obtained by high-throughput screening methods. Most important, the measurements obtained from proteins in their natural environment inside living cells provide the most physiologically relevant information about protein behavior currently available.
Acknowledgements T is research was supported in part by NIH P30DK079312 and the Indiana University School of Medicine. T e author thanks Dr. Yuansheng Sun and Dr. Jeff Liao (ISS Inc., Champaign, IL) for their advice and technical support and Michael Davidson for the plasmids encoding the FPs.
References [1] RN Day and MW Davidson , eds., T e Fluorescent Protein Revolution , CRC Press , Boca Raton , 2014 .
[2] A Periasamy and RN Day , eds., Molecular Imaging: FRET Microscopy and Spectroscopy . Oxford University Press , New York , 2005 .
[3] A Periasamy and RM Clegg , eds., FLIM Microscopy in Biology and Medicine . Taylor & Francis , Boca Raton , 2010 .
[4] R N Day , Methods 66 ( 2014 ) 200 – 07 . [5] DM Jameson et al ., Appl Spectrosc Rev 20 ( 1984 ) 55 – 106 .
[6] GI Redford and RM Clegg , J Fluoresc 15 ( 2005 ) 805 – 15 .
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[9] ML Markwardt et al ., PloS one 6 ( 2011 ) e17896 .
[10] J Goedhart et al ., Nat Methods 7 ( 2010 ) 137 – 39 .
Figure 6 : FRET-FLIM analysis of the heterologous interactions between the C/EBPα BZip domain and HP1α . (A) The intensity images for the nucleus of a cell expressing both the mTurquoise-BZip domain and mVenus-HP1α acquired in the acceptor channel, donor channel (the calibration bars are 10 μ m), and the corresponding lifetime map with I A / I D ratio indicated. (B) The intensity images for a cell expressing both the mTurquoiseN1 (localized throughout the cytoplasm and nucleus) and mVenus-HP1 α (nuclear) acquired in the acceptor channel, donor channel, and the corresponding lifetime map with the I A / I D ratio indicated. (C) FLIM was used to measure the donor lifetime in multiple ROI for each cell, and the FRET effi ciency (%) was determined. Each point represents the average E FRET (±SD) at the average I A / I D (±SD) for multiple ROI in individual cells expressing the indicated donor- and acceptor-labeled proteins. Reprinted from [ 4 ] with permission from Elsevier.
Conclusion T e FD FLIM method provides a direct measurement of FRET that requires no assumptions or corrections and is among the most accurate methods for monitoring protein interactions
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[11] AP Siegel et al ., J Biomed Opt 18 ( 2013 ) 25002 .
[12] C T aler et al ., Biophys J 89 ( 2005 ) 2736 – 49 .
[13] JR Lakowicz , Principles of Fluorescence Spectroscopy , 3rd Ed. , Springer , New York , 2006 .
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[15] C Stringari et al ., P Natl Acad Sci of the USA 108 ( 2011 ) 13582 – 87 .
[16] BK Wright et al ., Biophys J 103 ( 2012 ) L7 – 9 .
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