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  satFRET: estimation of Forster resonance energy transfer by acceptor saturation

Beutler, M., Makrogianneli, K., Vermeij, R. J., Keppler, M., Ng, T., Jovin, T. M., et al. (2008). satFRET: estimation of Forster resonance energy transfer by acceptor saturation. European Biophysics Journal with Biophysics Letters, 38(1), 69-82.

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 Creators:
Beutler, M.1, Author              
Makrogianneli, K., Author
Vermeij, R. J., Author
Keppler, M., Author
Ng, T., Author
Jovin, T. M., Author
Heintzmann, R., Author
Affiliations:
1Permanent Research Group Microsensor, Max Planck Institute for Marine Microbiology, Max Planck Society, ou_2481711              

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Free keywords: FRET; CFP; YFP; Excited state saturation; Fluorescence; Confocal microscopy; Sensitised emission; Photobleaching
 Abstract: We demonstrate theoretically and experimentally the quantification of Förster resonance energy transfer (FRET) by direct and systematic saturation of the excited state of acceptor molecules. This version of acceptor depletion methods for FRET estimation, denoted as “satFRET” is reversible and suitable for time-resolved measurements. The technique was investigated theoretically using the steady-state solution of the differential equation system of donor and acceptor molecular states. The influence of acceptor photobleaching during measurement was included in the model. Experimental verification was achieved with the FRET-pair Alexa 546- Alexa 633 loaded on particles in different stoichiometries and measured in a confocal microscope. Estimates of energy transfer efficiency by excited state saturation were compared to those obtained by measurements of sensitised emission and acceptor photobleaching. The results lead to a protocol that allows time-resolved FRET measurements of fixed and living cells on a conventional confocal microscope. This procedure was applied to fixed Chinese hamster ovary cells containing a cyan fluorescent protein and yellow fluorescent protein pair. The time resolution of the technique was demonstrated in a live T cell activation assay comparing the FRET efficiencies measured using a genetically encoded green and red fluorescent protein biosensor for GTP/GDP turnover to those measured by acceptor photobleaching of fixed cells.

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Language(s): eng - English
 Dates: 2008-09-042008-11
 Publication Status: Published in print
 Pages: 14
 Publishing info: -
 Table of Contents: -
 Rev. Type: Peer
 Identifiers: eDoc: 421146
ISI: 000260525000008
 Degree: -

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Title: European Biophysics Journal with Biophysics Letters
  Other : European Biophysics Journal with Biophysics Letters
Source Genre: Journal
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Publ. Info: Berlin : Springer
Pages: - Volume / Issue: 38 (1) Sequence Number: - Start / End Page: 69 - 82 Identifier: ISSN: 0175-7571
CoNE: https://pure.mpg.de/cone/journals/resource/954925487773