A Method to Quantify FRET Stoichiometry with Phasor Plot Analysis and Acceptor Lifetime Ingrowth
Schlachter, Simon C
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Chen, W., Avezov, E., Schlachter, S. C., Gielen, F., Laine, R., Harding, H., Hollfelder, F., et al. (2015). A Method to Quantify FRET Stoichiometry with Phasor Plot Analysis and Acceptor Lifetime Ingrowth. Biophysical Journal, 108 999-1002. https://doi.org/10.1016/j.bpj.2015.01.012
FRET is widely used for the study of protein-protein interactions in biological samples. However, it is difficult to quantify both the FRET efficiency (E) and the affinity (Kd) of the molecular interaction from intermolecular FRET signals in samples of unknown stoichiometry. Here, we present a method for the simultaneous quantification of the complete set of interaction parameters, including fractions of bound donors and acceptors, local protein concentrations, and dissociation constants, in each image pixel. The method makes use of fluorescence lifetime information from both donor and acceptor molecules and takes advantage of the linear properties of the phasor plot approach. We demonstrate the capability of our method in vitro in a microfluidic device and also in cells, via the determination of the binding affinity between tagged versions of glutathione and glutathione S-transferase, and via the determination of competitor concentration. The potential of the method is explored with simulations.
This work was funded by grants from the Medical Research Council, the Wellcome Trust, the Alzheimer Research UK Trust, and the Engineering and Physical Sciences Research Council. W.Y.C. is funded by a China Scholarship Council-Cambridge Scholarship. D.R. is a Principal Research Fellow of the Wellcome Trust.
Wellcome Trust (084812/Z/08/Z)
External DOI: https://doi.org/10.1016/j.bpj.2015.01.012
This record's URL: https://www.repository.cam.ac.uk/handle/1810/247206
Attribution 2.0 UK: England & Wales
Licence URL: http://creativecommons.org/licenses/by/2.0/uk/
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