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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.

Original publication

DOI

10.1002/jemt.21015

Type

Journal article

Journal

Microsc Res Tech

Publication Date

08/2011

Volume

74

Pages

788 - 793

Keywords

Cells, HeLa Cells, Humans, Luminescent Proteins, Protein Binding, Spectrometry, Fluorescence