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Variations on Negative Stain Electron Microscopy Methods: Tools for Tackling Challenging Systems

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 biologica...

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Autores principales: Scarff, Charlotte A., Fuller, Martin J. G., Thompson, Rebecca F., Iadaza, Matthew G.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MyJove Corporation 2018
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5912373/
https://www.ncbi.nlm.nih.gov/pubmed/29443097
http://dx.doi.org/10.3791/57199
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author Scarff, Charlotte A.
Fuller, Martin J. G.
Thompson, Rebecca F.
Iadaza, Matthew G.
author_facet Scarff, Charlotte A.
Fuller, Martin J. G.
Thompson, Rebecca F.
Iadaza, Matthew G.
author_sort Scarff, Charlotte A.
collection PubMed
description 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.
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spelling pubmed-59123732018-05-10 Variations on Negative Stain Electron Microscopy Methods: Tools for Tackling Challenging Systems Scarff, Charlotte A. Fuller, Martin J. G. Thompson, Rebecca F. Iadaza, Matthew G. J Vis Exp Biochemistry 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. MyJove Corporation 2018-02-06 /pmc/articles/PMC5912373/ /pubmed/29443097 http://dx.doi.org/10.3791/57199 Text en Copyright © 2018, Journal of Visualized Experiments http://creativecommons.org/licenses/by/3.0/us/ This is an open-access article distributed under the terms of the Creative Commons Attribution 3.0 License. To view a copy of this license, visithttp://creativecommons.org/licenses/by/3.0/us/
spellingShingle Biochemistry
Scarff, Charlotte A.
Fuller, Martin J. G.
Thompson, Rebecca F.
Iadaza, Matthew G.
Variations on Negative Stain Electron Microscopy Methods: Tools for Tackling Challenging Systems
title Variations on Negative Stain Electron Microscopy Methods: Tools for Tackling Challenging Systems
title_full Variations on Negative Stain Electron Microscopy Methods: Tools for Tackling Challenging Systems
title_fullStr Variations on Negative Stain Electron Microscopy Methods: Tools for Tackling Challenging Systems
title_full_unstemmed Variations on Negative Stain Electron Microscopy Methods: Tools for Tackling Challenging Systems
title_short Variations on Negative Stain Electron Microscopy Methods: Tools for Tackling Challenging Systems
title_sort variations on negative stain electron microscopy methods: tools for tackling challenging systems
topic Biochemistry
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5912373/
https://www.ncbi.nlm.nih.gov/pubmed/29443097
http://dx.doi.org/10.3791/57199
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