Scarff, CA orcid.org/0000-0001-6168-0060, Fuller, MJG, Thompson, RF et al. (1 more author) (2018) Variations on Negative Stain Electron Microscopy Methods: Tools for Tackling Challenging Systems. Journal of Visualized Experiments (132). e57199.
Abstract
Negative stain electron microscopy (EM) allows relatively simple and quick observation of macromolecules and macromolecular complexes through the use of contrast enhancing stain reagent. Although limited in resolution to a maximum of ~18 - 20 Å, negative stain EM is useful for a variety of biological problems and also provides a rapid means of assessing samples for cryo-electron microscopy (cryo-EM). The negative stain workflow is straightforward method; the sample is adsorbed onto a substrate, then a stain is applied, blotted, and dried to produce a thin layer of electron dense stain in which the particles are embedded. Individual samples can, however, behave in markedly different ways under varying staining conditions. This has led to the development of a large variety of substrate preparation techniques, negative staining reagents, and grid washing and blotting techniques. Determining the most appropriate technique for each individual sample must be done on a case-by-case basis and a microscopist must have access to a variety of different techniques to achieve the highest-quality negative stain results. Detailed protocols for two different substrate preparation methods and three different blotting techniques are provided, and an example of a sample that shows markedly different results depending on the method used is shown. In addition, the preparation of some common negative staining reagents, and two novel Lanthanide-based stains, is described with discussion regarding the use of each.
Metadata
Item Type: | Article |
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Authors/Creators: |
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Copyright, Publisher and Additional Information: | © 2018. This is an open access article. Licensed under a Creative Commons Attribution 3.0 License. |
Keywords: | Biochemistry, Issue 132, Electron microscopy, Negative stain, Biological TEM, Blotting, Electron microscopy grids, Carbon coating |
Dates: |
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Institution: | The University of Leeds |
Academic Units: | The University of Leeds > Faculty of Biological Sciences (Leeds) > School of Molecular and Cellular Biology (Leeds) > Cryo EM, Image Processing (Leeds) The University of Leeds > Faculty of Biological Sciences (Leeds) > School of Molecular and Cellular Biology (Leeds) > Structural Molecular Biology (Leeds) |
Funding Information: | Funder Grant number Wellcome Trust 204825/Z/16/Z |
Depositing User: | Symplectic Publications |
Date Deposited: | 19 Feb 2018 15:54 |
Last Modified: | 29 Jun 2020 11:26 |
Status: | Published |
Publisher: | Journal of Visualized Experiments (JoVE) |
Identification Number: | 10.3791/57199 |
Related URLs: | |
Open Archives Initiative ID (OAI ID): | oai:eprints.whiterose.ac.uk:127624 |