Chapter title |
Quantitative study of protein-protein interactions in live cell by dual-color fluorescence correlation spectroscopy.
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Chapter number | 31 |
Book title |
Fluorescence Spectroscopy and Microscopy
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Published in |
Methods in molecular biology, January 2014
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DOI | 10.1007/978-1-62703-649-8_31 |
Pubmed ID | |
Book ISBNs |
978-1-62703-648-1, 978-1-62703-649-8
|
Authors |
Sergi Padilla-Parra, Nicolas Audugé, Maïté Coppey-Moisan, Marc Tramier, Padilla-Parra, Sergi, Audugé, Nicolas, Coppey-Moisan, Maïté, Tramier, Marc |
Abstract |
Dual-color FCS is a powerful method to monitor protein-protein interactions in living cells. The main idea is based on the cross-correlation analysis of temporal fluorescence intensity fluctuations of two fluorescent proteins to obtain their co-diffusion and relative concentration. But, when performing these experiments, the spectral overlap in the emission of the two colors produces an artifact that corrupts the cross-correlation data: spectral bleed-through. We have shown that problems with cross talk are overcome with Fluorescence Lifetime Correlation Spectroscopy (FLCS). FLCS applied to dual-color cross-correlation, utilizing for example eGFP and mCherry fluorescent proteins, allows the determination of protein-protein interactions in living cells without the need of spectral bleed-through calibration. Here, we present in detail how this methodology can be implemented using a commercial setup (Microtime from PicoQuant, SP8 SMD from Leica or any conventional confocal with PicoQuant TCSPC module, and also with a Becker and Hickl TCSPC module). The dual-color FLCS experimental procedure where the different laser intensities do not have to be controlled during the experiment constitutes a very powerful technique to quantitatively study protein interactions in live samples. |
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