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Cleavable Linker Incorporation into a Synthetic Dye‐Nanobody‐Fluorescent Protein Assembly: FRET, FLIM and STED Microscopy

Von Wiley-VCH zur Verfügung gestellt

Conjugate and cleave? No paradox: compare! The target protein fused with a FRET donor was labeled with a nanobody bearing a FRET acceptor (synthetic dye). Then the acceptor was cleaved off, and the fluorescence outputs before and after cleavage were compared. Here are the news from FRET, FLIM and STED microscopy involving an anti-GFP nanobody bound with a fluorophore via a disulfide bond.


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, confocal imaging, and superresolution STED microscopy.

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