Dual-color fluorescence lifetime correlation spectroscopy to quantify protein-protein interactions in live cell

Dual‐color fluorescence correlation spectroscopy is an interesting method to quantify protein interaction in living cells. But, when performing these experiments, one must compensate for a known spectral bleed through artifact that corrupts cross‐correlation data. In this article, problems with cros...

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Bibliographic Details
Published in:Microscopy research and technique 2011-08, Vol.74 (8), p.788-793
Main Authors: Padilla-Parra, Sergi, Audugé, Nicolas, Coppey-Moisan, Maïté, Tramier, Marc
Format: Article
Language:English
Subjects:
Bleeding
Calibration
Cells - chemistry
Cells - metabolism
Correlation
FCCS
FCS
FLCS
Fluorescence
HeLa Cells
Humans
Lasers
Luminescent Proteins - chemistry
Luminescent Proteins - genetics
Luminescent Proteins - metabolism
Protein Binding
Proteins
quantitative fluorescence microscopy
Spectra
Spectrometry, Fluorescence - instrumentation
Spectrometry, Fluorescence - methods
Spectroscopy
TCSPC
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Summary:Dual‐color fluorescence correlation spectroscopy is an interesting method to quantify protein interaction in living cells. But, when performing these experiments, one must compensate for a known spectral bleed through artifact that corrupts cross‐correlation data. In this article, problems with crosstalk were overcome with an approach based on fluorescence lifetime correlation spectroscopy (FLCS). We show that FLCS applied to dual‐color EGFP and mCherry cross‐correlation allows the determination of protein–protein interactions in living cells without the need of spectral bleed through calibration. The methodology was validated by using EGFP‐mCherry tandem in comparison with coexpressed EGFP and mCherry in live cell. The dual‐color FLCS experimental procedure where the different laser intensities do not have to be controlled during experiment is really very helpful to study quantitatively protein interactions in live sample. Microsc. Res. Tech. 2011. © 2011 Wiley‐Liss, Inc.
ISSN:1059-910X
1097-0029
1097-0029
DOI:10.1002/jemt.21015