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

Overview

Journal Chembiochem

Specialty Biochemistry

Date 2022 Jul 15

PMID 35838445

Authors

Ayse Aktalay

Flavien Ponsot

Mariano L Bossi

Vladimir N Belov

Stefan W Hell

Affiliations

Soon will be listed here.

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, confocal imaging, and superresolution STED microscopy.

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