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Novel calibration method for flow cytometric fluorescence resonance energy transfer measurements between visible fluorescent proteins
Author(s) -
Nagy Peter,
Bene László,
Hyun William C.,
Vereb György,
Braun Manuela,
Antz Christof,
Paysan Jacques,
Damjanovich Sándor,
Park John W.,
Szöllsi János
Publication year - 2005
Publication title -
cytometry part a
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.316
H-Index - 90
eISSN - 1552-4930
pISSN - 1552-4922
DOI - 10.1002/cyto.a.20164
Subject(s) - förster resonance energy transfer , fluorescence , green fluorescent protein , yellow fluorescent protein , flow cytometry , biophysics , fluorescent protein , chemistry , acceptor , biology , microbiology and biotechnology , biochemistry , physics , optics , gene , condensed matter physics
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. © 2005 International Society for Analytical Cytology