TY - CHAP
T1 - Quantitative Study of protein-protein interactions in live cell by dual-color fluorescence correlation spectroscopy
AU - Padilla-Parra, Sergi
AU - Audugé, Nicolas
AU - Coppey-Moisan, Maïté
AU - Tramier, Marc
PY - 2014
Y1 - 2014
N2 - 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.
AB - 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.
KW - FCCS
KW - FCS
KW - FLCS
KW - Quantitative fluorescence microscopy
KW - TCSPC
UR - http://www.scopus.com/inward/record.url?scp=84934443967&partnerID=8YFLogxK
U2 - 10.1007/978-1-62703-649-8_31
DO - 10.1007/978-1-62703-649-8_31
M3 - Chapter
C2 - 24108650
AN - SCOPUS:84934443967
SN - 9781627036481
T3 - Methods in Molecular Biology
SP - 683
EP - 698
BT - Fluorescence Spectroscopy and Microscopy
PB - Humana Press Inc
ER -