This is a protocol preprint. Preprints are preliminary reports that have not undergone peer review. They should not be considered conclusive, used to inform clinical practice, or referenced by the media as validated information.
Method Article
Direct Synthesis of Gold Nanoparticles on Cysteine-rich Tags in Yeast Cells
Wanzhong He
National Institute of Biological Sciences, Beijing, 102206, China
Corresponding Author
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This work is licensed under a CC BY 4.0 License. Read Full License
Declarations:
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
This is a list of supplementary files associated with this preprint. Click to download.
- supplement1.pdf(2020-09-25 21:01:24)
- Figure2YeastProtocolv2.pdf(2020-08-10 11:30:12)
- supplement2.pdf(2020-09-25 21:01:24)
- Figure1YeastProtocolv2.pdf(2020-08-10 11:30:12)
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Associated Publications
Method Article
Direct Synthesis of Gold Nanoparticles on Cysteine-rich Tags in Yeast Cells
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
Declarations:
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