In-Vitro Characterization of mCerulean3_mRuby3 as a Novel FRET Pair with Favorable Bleed-Through Characteristics
| Autor: | Anne Marowsky, Michael Arand, Kira Erismann-Ebner |
|---|---|
| Přispěvatelé: | University of Zurich, Arand, Michael |
| Rok vydání: | 2019 |
| Předmět: |
lcsh:Biotechnology
Clinical Biochemistry 10050 Institute of Pharmacology and Toxicology 610 Medicine & health 1308 Clinical Biochemistry Endoplasmic Reticulum Article 03 medical and health sciences FAMPIR lcsh:TP248.13-248.65 Protein Interaction Mapping Fluorescence Resonance Energy Transfer fluorescent protein Humans Emission spectrum Fluorescent Dyes 030304 developmental biology 0303 health sciences 3D fluorescence spectra Chemistry Endoplasmic reticulum 030302 biochemistry & molecular biology excitation Membrane Proteins General Medicine In vitro Autofluorescence HEK293 Cells Förster resonance energy transfer Membrane protein biological sciences Biophysics 570 Life sciences biology Signal intensity Linker |
| Zdroj: | Biosensors Biosensors, Vol 9, Iss 1, p 33 (2019) Volume 9 Issue 1 |
| ISSN: | 2079-6374 |
| DOI: | 10.3390/bios9010033 |
| Popis: | In previous studies, we encountered substantial problems using the CFP_YFP Fö rster 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. |
| Databáze: | OpenAIRE |
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