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. 2021 May 14;8:635548. doi: 10.3389/fmolb.2021.635548

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Copyright © 2021 Eckenstaler and Benndorf.

This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

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Differentiation of dimeric and trimeric interactions using fluorescence intensity-based measurements. (A) HEK293T cells transfected with either the FRET triplet constructs (TCY, CTY, TYC) or the FRET doublets with their respective missing fluorophore (TY&C, TC&Y, YC&T). Representative images show the fluorescence intensity of mTurquoise2, YPet and mCherry before bleaching (unbleached), after the first bleaching step (mCherry bleached) and after the second bleaching step (YPet bleached). (B) Average fluorescence intensity of mTurquoise2 (mTurq2; blue), YPet (green) and mCherry (red) normalized to the first image acquired (n = 6). After mCherry bleaching, the YPet fluorescence intensity increased in YC&T, TCY, CTY and TYC and the mTurquoise2 fluorescence intensity increased in TC&Y, TCY, CTY and TYC, only. There was no increase in YPet fluorescence intensity in TY&C and TC&Y samples and no increase in mTurquoise2 fluorescence intensity in TY&C and YC&T samples due to the absence of specific YC- and TC-FRET. After YPet bleaching, the mTurquoise2 fluorescence intensity drastically increased in TY&C, TCY, CTY and TYC, whereas the mTurquoise2 fluorescence intensity remained rather constant in TC&Y and YC&T samples due to the absence of specific TY-FRET. The fluorescence increase corresponded to the unquenching of the FRET donor fluorescence intensity after bleaching its respective FRET acceptor, thereby abolishing FRET between them. (C–E) FRET efficiencies calculated from the change in fluorescence intensity of mTurquoise2 [TC-related FRET, (C)] and YPet [YC-related FRET, (D)] after mCherry bleaching and the FRET efficiency obtained from the change of mTurquoise2 fluorescence after YPet bleaching [TY-related FRET, (E)] (n = 6). Threshold values set to discriminate interaction-specific FRET from non-specific FRET are indicated by gray lines. Note the different response patterns of FRET doublets compared to those from FRET triplets. (F) Color coded response pattern of fluorescence intensity-based measurements. A Yes-response (occurrence of specific FRET) is indicated by a green and a No-response (no or non-specific FRET) is indicated by a red square. According to these patterns, dimeric interactions (FRET doublets) can be clearly discriminated from trimeric interactions (FRET triplets).