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.
Tabata S, Jevtic M, Kurashige N, et al. Electron microscopic detection of single membrane proteins by a specific chemical labeling. iScience. 2019;22(12):256-268. doi:10.1016/j.isci.2019.11.025
Tabata, S., Jevtic, M., Kurashige, N., Fuchida, H., Kido, M., Tani, K., … Ojida, A. (2019). Electron microscopic detection of single membrane proteins by a specific chemical labeling. IScience. Elsevier. https://doi.org/10.1016/j.isci.2019.11.025
Tabata, Shigekazu, Marijo Jevtic, Nobutaka Kurashige, Hirokazu Fuchida, Munetsugu Kido, Kazushi Tani, Naoki Zenmyo, et al. “Electron Microscopic Detection of Single Membrane Proteins by a Specific Chemical Labeling.” IScience. Elsevier, 2019. https://doi.org/10.1016/j.isci.2019.11.025.
S. Tabata et al., “Electron microscopic detection of single membrane proteins by a specific chemical labeling,” iScience, vol. 22, no. 12. Elsevier, pp. 256–268, 2019.
Tabata S, Jevtic M, Kurashige N, Fuchida H, Kido M, Tani K, Zenmyo N, Uchinomiya S, Harada H, Itakura M, Hamachi I, Shigemoto R, Ojida A. 2019. Electron microscopic detection of single membrane proteins by a specific chemical labeling. iScience. 22(12), 256–268.
Tabata, Shigekazu, et al. “Electron Microscopic Detection of Single Membrane Proteins by a Specific Chemical Labeling.” IScience, vol. 22, no. 12, Elsevier, 2019, pp. 256–68, doi:10.1016/j.isci.2019.11.025.
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