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Method Article
Wanzhong He
National Institute of Biological Sciences, Beijing, 102206, China
Corresponding Author
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We developed a novel auto-nucleation suppressed mechanism (ANSM) for direct synthesis of EM-visible gold nanoparticles (AuNPs) on cysteine-rich tags (e.g., metallothionein) in cells for single-molecule detection with electron microscopy (it accompanies our Nature Method manuscript, Jiang et al. 2020 [1]). Both tagged-fusion proteins expressed in cells (e.g.bacteria, yeast and mammalian cells) and antigens stained with antibody-tag fusion proteins can be visualized by this protocol. Here we describe the typical protocols (both the chemical fixation and the high pressure freezing cases) developed for ANSM-based AuNP synthesis in yeast cells expressing metallothionein (MTn) tags (Figure 1). This approach should be useful for EM visualization of single-molecule in yeast cells, and easier adapted for bacterial cells.
Keywords
Cysteine-rich tag, electron microscopy, gold nanoparticle, yeast cell, Schizosaccharomyces pombe, metallothionein, AuNP synthesis, single-molecule chemical fixation, oxidization, high pressure freezing, freeze-substitution fixation, gold enhancement, imaging, ultrastructure, labeling
Figure 1
Figure 2
The National Institute of Biological Sciences, Beijing, filed a PCT patent (WO2019024707A1) covering part of the information contained in this article.
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Associated Publications
Method Article
Wanzhong He
National Institute of Biological Sciences, Beijing, 102206, China
Corresponding Author
Version History
CURRENT STATUS: Posted
Version 1
Posted 10 Aug, 2020
No community comments so far
Metrics
Comments: 0
HTML Views: ...
License:
This work is licensed under a CC BY 4.0 License. Read Full License
We developed a novel auto-nucleation suppressed mechanism (ANSM) for direct synthesis of EM-visible gold nanoparticles (AuNPs) on cysteine-rich tags (e.g., metallothionein) in cells for single-molecule detection with electron microscopy (it accompanies our Nature Method manuscript, Jiang et al. 2020 [1]). Both tagged-fusion proteins expressed in cells (e.g.bacteria, yeast and mammalian cells) and antigens stained with antibody-tag fusion proteins can be visualized by this protocol. Here we describe the typical protocols (both the chemical fixation and the high pressure freezing cases) developed for ANSM-based AuNP synthesis in yeast cells expressing metallothionein (MTn) tags (Figure 1). This approach should be useful for EM visualization of single-molecule in yeast cells, and easier adapted for bacterial cells.
Figure 1
Figure 2
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