Abstract
In previous studies, we encountered substantial problems using the CFP_YFP Förster resonance energy transfer (FRET) pair to analyze protein proximity in the endoplasmic reticulum of live cells. Bleed-through of the donor emission into the FRET channel and overlap of the FRET emission wavelength with highly variable cellular autofluorescence significantly compromised the sensitivity of our analyses. Here, we propose mCerulean3 and mRuby3 as a new FRET pair to potentially overcome these problems. Fusion of the two partners with a trypsin-cleavable linker allowed the direct comparison of the FRET signal characteristics of the associated partners with those of the completely dissociated partners. We compared our new FRET pair with the canonical CFP_YFP and the more recent mClover3_mRuby3 pairs and found that, despite a lower total FRET signal intensity, the novel pair had a significantly better signal to noise ratio due to lower donor emission bleed-through. This and the fact that the mRuby3 emission spectrum did not overlap with that of common cellular autofluorescence renders the mCerulean3_mRuby3 FRET pair a promising alternative to the common CFP_YFP FRET pair for the interaction analysis of membrane proteins in living cells.
Highlights
Förster resonance energy transfer (FRET) is the non-radiative transfer of energy from an excited donor fluorophore to a neighboring acceptor fluorophore by dipole-dipole coupling, resulting in the emission of fluorescence characteristic for the acceptor [1]
Since the development of respective fluorescent protein probes [2], FRET has become an important analytical strategy to analyze the association of target proteins in live cells [3]
We have recently developed FAMPIR (FRET analysis of membrane protein interaction in the endoplasmic reticulum), a FACS-based FRET procedure to analyze the association of membrane proteins in the endoplasmic reticulum of the mammalian cell line HEK293 [4]
Summary
Förster resonance energy transfer (FRET) is the non-radiative transfer of energy from an excited donor fluorophore to a neighboring acceptor fluorophore by dipole-dipole coupling, resulting in the emission of fluorescence characteristic for the acceptor [1]. Since the development of respective fluorescent protein probes [2], FRET has become an important analytical strategy to analyze the association of target proteins in live cells [3]. We have recently developed FAMPIR (FRET analysis of membrane protein interaction in the endoplasmic reticulum), a FACS-based FRET procedure to analyze the association of membrane proteins in the endoplasmic reticulum of the mammalian cell line HEK293 [4]. FAMPIR uses the canonical CFP_YFP FRET pair for the analysis of protein association. In the range of the emission spectrum of the FRET acceptor YFP, Biosensors 2019, 9, 33; doi:10.3390/bios9010033 www.mdpi.com/journal/biosensors
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