Header

UZH-Logo

Maintenance Infos

In-Vitro Characterization of mCerulean3_mRuby3 as a Novel FRET Pair with Favorable Bleed-Through Characteristics


Erismann-Ebner, Kira; Marowsky, Anne; Arand, Michael (2019). In-Vitro Characterization of mCerulean3_mRuby3 as a Novel FRET Pair with Favorable Bleed-Through Characteristics. Biosensors, 9:33.

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.

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.

Statistics

Citations

Altmetrics

Downloads

4 downloads since deposited on 29 Oct 2019
4 downloads since 12 months
Detailed statistics

Additional indexing

Item Type:Journal Article, refereed, original work
Communities & Collections:04 Faculty of Medicine > Institute of Pharmacology and Toxicology
07 Faculty of Science > Institute of Pharmacology and Toxicology
Dewey Decimal Classification:570 Life sciences; biology
610 Medicine & health
Language:English
Date:28 February 2019
Deposited On:29 Oct 2019 14:21
Last Modified:01 Nov 2019 13:45
Publisher:MDPI Publishing
ISSN:2079-6374
OA Status:Gold
Free access at:PubMed ID. An embargo period may apply.
Publisher DOI:https://doi.org/10.3390/bios9010033
PubMed ID:30823443

Download

Gold Open Access

Download PDF  'In-Vitro Characterization of mCerulean3_mRuby3 as a Novel FRET Pair with Favorable Bleed-Through Characteristics'.
Preview
Content: Published Version
Filetype: PDF
Size: 2MB
View at publisher
Licence: Creative Commons: Attribution 4.0 International (CC BY 4.0)