Novel calibration method for flow cytometric fluorescence resonance energy transfer measurements between visible fluorescent proteins
Article first published online: 14 SEP 2005
Copyright © 2005 Wiley-Liss, Inc.
Cytometry Part A
Special Issue: Honoring the Lifetime Achievements of Mack J. Fulwyler
Volume 67A, Issue 2, pages 86–96, October 2005
How to Cite
Nagy, P., Bene, L., Hyun, W. C., Vereb, G., Braun, M., Antz, C., Paysan, J., Damjanovich, S., Park, J. W. and Szöllsi, J. (2005), Novel calibration method for flow cytometric fluorescence resonance energy transfer measurements between visible fluorescent proteins. Cytometry, 67A: 86–96. doi: 10.1002/cyto.a.20164
- Issue published online: 20 SEP 2005
- Article first published online: 14 SEP 2005
- Manuscript Accepted: 31 MAR 2005
- Manuscript Revised: 8 MAR 2005
- Manuscript Received: 11 JAN 2005
- Hungarian Ministry of Health. Grant Numbers: ETT524/2003, 532/2003
- Hungarian Academy of Sciences. Grant Numbers: OTKA T043061, F049025, T037831
- European Union. Grant Number: LSHB-CT-2004-503467
- Fogarty International Collaboration Award. Grant Number: 1 R02 TW00871-01A2
- Békésy Fellowship, Hungarian Ministry of Education
- flow cytometry;
- fluorescence resonance energy transfer;
- green fluorescent protein
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.
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.
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.
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