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A small protein probe for correlated microscopy of endogenous proteins

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Title: A small protein probe for correlated microscopy of endogenous proteins
Authors: Marit A. de Beer, Jeroen Kuipers, Paul M. P. van Bergen en Henegouwen, Ben N. G. Giepmans
Contributors: Sub Cell Biology, Cell Biology, Neurobiology and Biophysics
Source: Histochem Cell Biol
Publisher Information: Springer Science and Business Media LLC, 2018.
Publication Year: 2018
Subject Terms: NANOBODIES, 0301 basic medicine, Fluorescent Dyes/analysis, TOOLBOX, Cells, Peroxidase/analysis, Green Fluorescent Proteins, Microscopy, Electron/methods, Fluorescence/methods, QUANTUM DOTS, 03 medical and health sciences, FLUORESCENT PROTEIN, Journal Article, Humans, Microscopy, Fluorescence/methods, Cells, Cultured, Fluorescent Dyes, Peroxidase, ELECTRON-MICROSCOPY, Microscopy, Original Paper, 0303 health sciences, Cultured, Electron/methods, Microscopy, Electron, LIGHT, Microscopy, Fluorescence, Green Fluorescent Proteins/chemistry, ANTIBODIES, CELLS, VISUALIZATION, EPIDERMAL-GROWTH-FACTOR
Description: 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.
Document Type: Article
Other literature type
File Description: image/pdf
Language: English
ISSN: 1432-119X
0948-6143
DOI: 10.1007/s00418-018-1632-6
Access URL: https://link.springer.com/content/pdf/10.1007/s00418-018-1632-6.pdf
https://pubmed.ncbi.nlm.nih.gov/29327239
https://research-portal.uu.nl/en/publications/875079f7-5380-4435-b859-5275bbfcad80
https://doi.org/10.1007/s00418-018-1632-6
https://dspace.library.uu.nl/bitstream/1874/377569/1/small.pdf
https://www.narcis.nl/publication/RecordID/oai%3Apure.rug.nl%3Apublications%2Fbf332740-7d6a-4a6e-aaf2-11bcc0631c87
https://core.ac.uk/display/158765546
https://link.springer.com/article/10.1007/s00418-018-1632-6
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5816110
https://www.rug.nl/research/portal/publications/a-small-protein-probe-for-correlated-microscopy-of-endogenous-proteins(bf332740-7d6a-4a6e-aaf2-11bcc0631c87).html
https://dspace.library.uu.nl/handle/1874/377569
Rights: CC BY
Accession Number: edsair.doi.dedup.....8e98f1dbb30a96f11c5f8a36a79069a0
Database: OpenAIRE
Description
Abstract: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.
ISSN:1432119X
09486143
DOI:10.1007/s00418-018-1632-6