可裂解连接子在合成染料-纳米抗体-荧光蛋白组装体中的应用：FRET、FLIM 和 STED 显微镜。
Cleavable Linker Incorporation into a Synthetic Dye-Nanobody-Fluorescent Protein Assembly: FRET, FLIM and STED Microscopy.
机构信息
Department of Optical Nanoscopy, Max Planck Institute for Medical Research (MPI-MR), Jahnstraße 29, 69120, Heidelberg, Germany.
Department of NanoBiophotonics, Max Planck Institute for Multidisciplinary Sciences (MPI-NAT), Am Fassberg 11, 37077, Göttingen, Germany.
出版信息
Chembiochem. 2022 Sep 16;23(18):e202200395. doi: 10.1002/cbic.202200395. Epub 2022 Aug 16.
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.
一种带有二硫键（S-S）连接物和马来酰亚胺基团（Rho594-S2-mal）的明亮且光稳定的荧光染料，作为可切割和反应性位点，被合成并与抗 GFP 纳米抗体（NB）缀合。在体外和细胞内研究了带有一个或两个 Rho594-S2-mal 残基的抗 GFP NB 标记的 EGFP（FRET 供体）与 Rho594-S2-mal 的结合。用二硫苏糖醇（DTT）切割连接物，恢复供体（FP）信号。生物缀合物（FP-NB-dye）被应用于 FRET-FLIM 测定、共聚焦成像和超分辨率 STED 显微镜。
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