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Journal Article

Cleavable linker incorporation into a synthetic dye - nanobody -fluorescent protein assembly: FRET, FLIM and STED microscopy

MPS-Authors
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Aktalay,  Ayse
Optical Nanoscopy, Max Planck Institute for Medical Research, Max Planck Society;

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Bossi,  Mariano L.
Optical Nanoscopy, Max Planck Institute for Medical Research, Max Planck Society;

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Hell,  Stefan W.
Optical Nanoscopy, Max Planck Institute for Medical Research, Max Planck Society;

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https://chemistry-europe.onlinelibrary.wiley.com/doi/10.1002/cbic.202200395
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https://chemistry-europe.onlinelibrary.wiley.com/doi/epdf/10.1002/cbic.202200395
(Any fulltext)

https://chemistry-europe.onlinelibrary.wiley.com/action/downloadSupplement?doi=10.1002%2Fcbic.202200395&file=cbic202200395-s1-Sup_Inf_accepted.pdf
(Supplementary material)

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Citation

Aktalay, A., Ponsot, F., Bossi, M. L., Belov, V. N., & Hell, S. W. (2022). Cleavable linker incorporation into a synthetic dye - nanobody -fluorescent protein assembly: FRET, FLIM and STED microscopy. Chembiochem, 23(18): e202200395, pp. 1-8. doi:10.1002/cbic.202200395.


Cite as: https://hdl.handle.net/21.11116/0000-000A-CF65-C
Abstract
A bright and photostable fluorescent dye with a disulfide (S-S) linker and maleimide group (Rho594-S2-mal), as cleavable and reactive sites, was synthesized and conjugated with anti-GFP nanobodies (NB). The binding of EGFP (FRET donor) with anti-GFP NB labeled with one or two Rho594-S2-mal residues was studied in vitro and in cellulo. The linker was cleaved with dithiothreitol recovering the donor (FP) signal. The bioconjugates (FP-NB-dye) were applied in FRET-FLIM assays, multiplexed confocal imaging, and superresolution STED microscopy.