Novel calibration method for flow cytometric fluorescence resonance energy transfer measurements between visible fluorescent proteins

Peter Nagy, László Bene, William C. Hyun, György Vereb, Manuela Braun, Christof Antz, Jacques Paysan, Sándor Damjanovich, John W. Park, János Szöllosi

Research output: Contribution to journalArticle

27 Citations (Scopus)


Background: The combination of fluorescence resonance energy transfer (FRET) and flow cytometry offers a statistically firm approach to study protein associations. Fusing green fluorescent protein (GFP) to a studied protein usually does not disturb the normal function of a protein, but quantitation of FRET efficiency calculated between GFP derivatives poses a problem in flow cytometry. Methods: We generated chimeras in which cyan fluorescent protein (CFP) was separated by amino acid linkers of different sizes from yellow fluorescent protein (YFP) and used them to calibrate the cell-by-cell flow cytometric FRET measurements carried out on two different dual-laser flow cytometers. Then, CFP-Kip1 was coexpressed in yeast cells with YFP and cyclin-dependent kinase-2 (Cdk2) and served as a positive control for FRET measurements, and CFP-Kip1 coexpressed with a random peptide fused to YFP was the negative control. Results: We measured donor, direct, and sensitized acceptor fluorescence intensities and developed a novel way to calculate a factor (α) that characterized the fluorescence intensity of acceptor molecules relative to the same number of excited donor molecules, which is essential for quantifying FRET efficiency. This was achieved by calculating FRET efficiency in two different ways and minimizing the squared difference between the two results by changing α. Our method reliably detected the association of Cdk2 with its inhibitor, Kip1, whereas the nonspecific FRET efficiency between Cdk2 and a random peptide was negligible. We identified and sorted subpopulations of yeast cells showing interaction between the studied proteins. Conclusions: We have described a straightforward novel calibration method to accurately quantitate FRET efficiency between GFP derivatives in flow cytometry.

Original languageEnglish
Pages (from-to)86-96
Number of pages11
JournalCytometry Part A
Issue number2
Publication statusPublished - Oct 1 2005


  • Flow cytometry
  • Fluorescence resonance energy transfer
  • Green fluorescent protein

ASJC Scopus subject areas

  • Pathology and Forensic Medicine
  • Histology
  • Cell Biology

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