22 February 2013 Single molecule FRET using the FRET pair DRONPA/PhotoActivable mCherry
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Abstract
Photoswitchable and photoactivable proteins Dronpa and PhotoActivable mCherry (PA-mCherry) respectively, were used in order to perform FRET (Förster Resonance Energy Transfer) imaging at the single molecule level, using a FRET standard construct consisting of Dronpa and PA-mCherry separated by seven amino acids expressed in cells. Given Dronpa’s complex photophysical properties and the existence of a preswitched emissive state, irradiation conditions at 491 and 405 nm were optimised. We discuss strategies for observing FRET at the single molecule level with photoactivatable proteins by monitoring modifications in the donor and acceptors emissive states.
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Viviane Devauges, Viviane Devauges, Elena Ortiz-Zapater, Elena Ortiz-Zapater, Christina Efthymiou, Christina Efthymiou, Melanie Keppler, Melanie Keppler, Jody Barbeau, Jody Barbeau, Daniel R. Matthews, Daniel R. Matthews, James Monypenny of Pitmilly, James Monypenny of Pitmilly, Paul R. Barber, Paul R. Barber, Daniel Rolfe, Daniel Rolfe, Tony Ng, Tony Ng, Simon M. Ameer-Beg, Simon M. Ameer-Beg, } "Single molecule FRET using the FRET pair DRONPA/PhotoActivable mCherry", Proc. SPIE 8590, Single Molecule Spectroscopy and Superresolution Imaging VI, 85900A (22 February 2013); doi: 10.1117/12.2002470; https://doi.org/10.1117/12.2002470
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