Suspending samples over carbon holey films increases heterogeneity of molecular orientations in negative stain electron microscopy
Negative stain electron microscopy (NSEM) is a simple and cost effective method to analyze a wide variety of specimens, especially proteins. In traditional NSEM, the protein sample is applied to and supported by a continuous carbon film. Unfortunately, many proteins stick to the carbon film with a l...
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doaj-3240870d4cc24063855c2a3e0a57992a2020-11-25T01:30:23ZengElsevierMethodsX2215-01612019-01-016582586Suspending samples over carbon holey films increases heterogeneity of molecular orientations in negative stain electron microscopyKatayoun Mansouri0Robert J. Edwards1Duke Human Vaccine Institute, Duke University, Durham, NC, United StatesCorresponding author.; Duke Human Vaccine Institute, Duke University, Durham, NC, United StatesNegative stain electron microscopy (NSEM) is a simple and cost effective method to analyze a wide variety of specimens, especially proteins. In traditional NSEM, the protein sample is applied to and supported by a continuous carbon film. Unfortunately, many proteins stick to the carbon film with a limited number of orientations. Because the restricted orientation limits the available views of the molecule, information about the three-dimensional structure of the molecule is likewise limited. The method presented here overcomes this limitation by using a carbon holey film combined with 1-octadecanol as a spreading agent. We demonstrate this method with solubilized envelope (Env) proteins from HIV, which typically show a restricted orientation on continuous carbon film, and show the following: • 1-octadecanol added to negative stain aids the formation of a continuous sample-stain layer spanning the holes of a holey carbon film. • Samples negatively stained over holes show less restricted orientation, resulting in better single particle reconstructions. Method name: Negative stain over holes, Keywords: 1-Octadecanol, Uranyl formate, Carbon film, Preferred orientation, Single particle reconstructionshttp://www.sciencedirect.com/science/article/pii/S2215016119300433 |
collection |
DOAJ |
language |
English |
format |
Article |
sources |
DOAJ |
author |
Katayoun Mansouri Robert J. Edwards |
spellingShingle |
Katayoun Mansouri Robert J. Edwards Suspending samples over carbon holey films increases heterogeneity of molecular orientations in negative stain electron microscopy MethodsX |
author_facet |
Katayoun Mansouri Robert J. Edwards |
author_sort |
Katayoun Mansouri |
title |
Suspending samples over carbon holey films increases heterogeneity of molecular orientations in negative stain electron microscopy |
title_short |
Suspending samples over carbon holey films increases heterogeneity of molecular orientations in negative stain electron microscopy |
title_full |
Suspending samples over carbon holey films increases heterogeneity of molecular orientations in negative stain electron microscopy |
title_fullStr |
Suspending samples over carbon holey films increases heterogeneity of molecular orientations in negative stain electron microscopy |
title_full_unstemmed |
Suspending samples over carbon holey films increases heterogeneity of molecular orientations in negative stain electron microscopy |
title_sort |
suspending samples over carbon holey films increases heterogeneity of molecular orientations in negative stain electron microscopy |
publisher |
Elsevier |
series |
MethodsX |
issn |
2215-0161 |
publishDate |
2019-01-01 |
description |
Negative stain electron microscopy (NSEM) is a simple and cost effective method to analyze a wide variety of specimens, especially proteins. In traditional NSEM, the protein sample is applied to and supported by a continuous carbon film. Unfortunately, many proteins stick to the carbon film with a limited number of orientations. Because the restricted orientation limits the available views of the molecule, information about the three-dimensional structure of the molecule is likewise limited. The method presented here overcomes this limitation by using a carbon holey film combined with 1-octadecanol as a spreading agent. We demonstrate this method with solubilized envelope (Env) proteins from HIV, which typically show a restricted orientation on continuous carbon film, and show the following: • 1-octadecanol added to negative stain aids the formation of a continuous sample-stain layer spanning the holes of a holey carbon film. • Samples negatively stained over holes show less restricted orientation, resulting in better single particle reconstructions. Method name: Negative stain over holes, Keywords: 1-Octadecanol, Uranyl formate, Carbon film, Preferred orientation, Single particle reconstructions |
url |
http://www.sciencedirect.com/science/article/pii/S2215016119300433 |
work_keys_str_mv |
AT katayounmansouri suspendingsamplesovercarbonholeyfilmsincreasesheterogeneityofmolecularorientationsinnegativestainelectronmicroscopy AT robertjedwards suspendingsamplesovercarbonholeyfilmsincreasesheterogeneityofmolecularorientationsinnegativestainelectronmicroscopy |
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