A small protein probe for correlated microscopy of endogenous proteins

Autor: de Beer, Marit A, Kuipers, Jeroen, van Bergen En Henegouwen, Paul M P, Giepmans, Ben N G, Sub Cell Biology, Celbiologie
Přispěvatelé: Center for Liver, Digestive and Metabolic Diseases (CLDM), ​Basic and Translational Research and Imaging Methodology Development in Groningen (BRIDGE), Sub Cell Biology, Celbiologie
Jazyk: angličtina
Rok vydání: 2018
Předmět:
0301 basic medicine
APEX2
NANOBODIES
Fluorescent Dyes/analysis
Peroxidase/analysis
QUANTUM DOTS
Microscopy
Electron/methods
Green fluorescent protein
Microscopy
Correlated microscopy
Cells
Cultured
ELECTRON-MICROSCOPY
Cultured
Chemistry
Resolution (electron density)
FLIPPER
Electron/methods
Fluorescence
Medical Laboratory Technology
LIGHT
EPIDERMAL-GROWTH-FACTOR
Histology
TOOLBOX
Green Fluorescent Proteins
Context (language use)
Fluorescence/methods
03 medical and health sciences
FLUORESCENT PROTEIN
Journal Article
Humans
Molecular Biology
Microscopy
Fluorescence/methods
Fluorescent Dyes
Peroxidase
Original Paper
Cell Biology
Fusion protein
Microscopy
Electron
030104 developmental biology
Microscopy
Fluorescence
Green Fluorescent Proteins/chemistry
ANTIBODIES
CELLS
Ultrastructure
Biophysics
Nanobody
VISUALIZATION
Ectopic expression
Probes
Zdroj: Histochemistry and Cell Biology, 149(3), 261. Springer Verlag
Histochemistry and cell biology, 149(3), 261-268. SPRINGER
Histochemistry and Cell Biology
ISSN: 0948-6143
Popis: Probes are essential to visualize proteins in their cellular environment, both using light microscopy as well as electron microscopy (EM). Correlated light microscopy and electron microscopy (CLEM) requires probes that can be imaged simultaneously by both optical and electron-dense signals. Existing combinatorial probes often have impaired efficiency, need ectopic expression as a fusion protein, or do not target endogenous proteins. Here, we present FLIPPER-bodies to label endogenous proteins for CLEM. Fluorescent Indicator and Peroxidase for Precipitation with EM Resolution (FLIPPER), the combination of a fluorescent protein and a peroxidase, is fused to a nanobody against a target of interest. The modular nature of these probes allows an easy exchange of components to change its target or color. A general FLIPPER-body targeting GFP highlights histone2B-GFP both in fluorescence and in EM. Similarly, endogenous EGF receptors and HER2 are visualized at nm-scale resolution in ultrastructural context. The small and flexible FLIPPER-body outperforms IgG-based immuno-labeling, likely by better reaching the epitopes. Given the modular domains and possibilities of nanobody generation for other targets, FLIPPER-bodies have high potential to become a universal tool to identify proteins in immuno-CLEM with increased sensitivity compared to current approaches. Electronic supplementary material The online version of this article (10.1007/s00418-018-1632-6) contains supplementary material, which is available to authorized users.
Databáze: OpenAIRE
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