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Electron Microscopic Detection of Single Membrane Proteins by a Specific Chemical Labeling

Electron microscopy (EM) is a technology that enables visualization of single proteins at a nanometer resolution. However, current protein analysis by EM mainly relies on immunolabeling with gold-particle-conjugated antibodies, which is compromised by large size of antibody, precluding precise detec...

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Autores principales: Tabata, Shigekazu, Jevtic, Marijo, Kurashige, Nobutaka, Fuchida, Hirokazu, Kido, Munetsugu, Tani, Kazushi, Zenmyo, Naoki, Uchinomiya, Shohei, Harada, Harumi, Itakura, Makoto, Hamachi, Itaru, Shigemoto, Ryuichi, Ojida, Akio
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Elsevier 2019
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6906691/
https://www.ncbi.nlm.nih.gov/pubmed/31786521
http://dx.doi.org/10.1016/j.isci.2019.11.025
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author Tabata, Shigekazu
Jevtic, Marijo
Kurashige, Nobutaka
Fuchida, Hirokazu
Kido, Munetsugu
Tani, Kazushi
Zenmyo, Naoki
Uchinomiya, Shohei
Harada, Harumi
Itakura, Makoto
Hamachi, Itaru
Shigemoto, Ryuichi
Ojida, Akio
author_facet Tabata, Shigekazu
Jevtic, Marijo
Kurashige, Nobutaka
Fuchida, Hirokazu
Kido, Munetsugu
Tani, Kazushi
Zenmyo, Naoki
Uchinomiya, Shohei
Harada, Harumi
Itakura, Makoto
Hamachi, Itaru
Shigemoto, Ryuichi
Ojida, Akio
author_sort Tabata, Shigekazu
collection PubMed
description Electron microscopy (EM) is a technology that enables visualization of single proteins at a nanometer resolution. However, current protein analysis by EM mainly relies on immunolabeling with gold-particle-conjugated antibodies, which is compromised by large size of antibody, precluding precise detection of protein location in biological samples. Here, we develop a specific chemical labeling method for EM detection of proteins at single-molecular level. Rational design of α-helical peptide tag and probe structure provided a complementary reaction pair that enabled specific cysteine conjugation of the tag. The developed chemical labeling with gold-nanoparticle-conjugated probe showed significantly higher labeling efficiency and detectability of high-density clusters of tag-fused G protein-coupled receptors in freeze-fracture replicas compared with immunogold labeling. Furthermore, in ultrathin sections, the spatial resolution of the chemical labeling was significantly higher than that of antibody-mediated labeling. These results demonstrate substantial advantages of the chemical labeling approach for single protein visualization by EM.
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spelling pubmed-69066912019-12-20 Electron Microscopic Detection of Single Membrane Proteins by a Specific Chemical Labeling Tabata, Shigekazu Jevtic, Marijo Kurashige, Nobutaka Fuchida, Hirokazu Kido, Munetsugu Tani, Kazushi Zenmyo, Naoki Uchinomiya, Shohei Harada, Harumi Itakura, Makoto Hamachi, Itaru Shigemoto, Ryuichi Ojida, Akio iScience Article Electron microscopy (EM) is a technology that enables visualization of single proteins at a nanometer resolution. However, current protein analysis by EM mainly relies on immunolabeling with gold-particle-conjugated antibodies, which is compromised by large size of antibody, precluding precise detection of protein location in biological samples. Here, we develop a specific chemical labeling method for EM detection of proteins at single-molecular level. Rational design of α-helical peptide tag and probe structure provided a complementary reaction pair that enabled specific cysteine conjugation of the tag. The developed chemical labeling with gold-nanoparticle-conjugated probe showed significantly higher labeling efficiency and detectability of high-density clusters of tag-fused G protein-coupled receptors in freeze-fracture replicas compared with immunogold labeling. Furthermore, in ultrathin sections, the spatial resolution of the chemical labeling was significantly higher than that of antibody-mediated labeling. These results demonstrate substantial advantages of the chemical labeling approach for single protein visualization by EM. Elsevier 2019-11-16 /pmc/articles/PMC6906691/ /pubmed/31786521 http://dx.doi.org/10.1016/j.isci.2019.11.025 Text en © 2019 The Author(s) http://creativecommons.org/licenses/by/4.0/ This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
Tabata, Shigekazu
Jevtic, Marijo
Kurashige, Nobutaka
Fuchida, Hirokazu
Kido, Munetsugu
Tani, Kazushi
Zenmyo, Naoki
Uchinomiya, Shohei
Harada, Harumi
Itakura, Makoto
Hamachi, Itaru
Shigemoto, Ryuichi
Ojida, Akio
Electron Microscopic Detection of Single Membrane Proteins by a Specific Chemical Labeling
title Electron Microscopic Detection of Single Membrane Proteins by a Specific Chemical Labeling
title_full Electron Microscopic Detection of Single Membrane Proteins by a Specific Chemical Labeling
title_fullStr Electron Microscopic Detection of Single Membrane Proteins by a Specific Chemical Labeling
title_full_unstemmed Electron Microscopic Detection of Single Membrane Proteins by a Specific Chemical Labeling
title_short Electron Microscopic Detection of Single Membrane Proteins by a Specific Chemical Labeling
title_sort electron microscopic detection of single membrane proteins by a specific chemical labeling
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6906691/
https://www.ncbi.nlm.nih.gov/pubmed/31786521
http://dx.doi.org/10.1016/j.isci.2019.11.025
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