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Title: Optimized Negative Staining: a High-throughput Protocol for Examining Small and Asymmetric Protein Structure by Electron Microscopy

Abstract

Structural determination of proteins is rather challenging for proteins with molecular masses between 40 - 200 kDa. Considering that more than half of natural proteins have a molecular mass between 40 - 200 kDa, a robust and high-throughput method with a nanometer resolution capability is needed. Negative staining (NS) electron microscopy (EM) is an easy, rapid, and qualitative approach which has frequently been used in research laboratories to examine protein structure and protein-protein interactions. Unfortunately, conventional NS protocols often generate structural artifacts on proteins, especially with lipoproteins that usually form presenting rouleaux artifacts. By using images of lipoproteins from cryo-electron microscopy (cryo-EM) as a standard, the key parameters in NS specimen preparation conditions were recently screened and reported as the optimized NS protocol (OpNS), a modified conventional NS protocol. Artifacts like rouleaux can be greatly limited by OpNS, additionally providing high contrast along with reasonably high-resolution (near 1 nm) images of small and asymmetric proteins. These high-resolution and high contrast images are even favorable for an individual protein (a single object, no average) 3D reconstruction, such as a 160 kDa antibody, through the method of electron tomography. Moreover, OpNS can be a high-throughput tool to examine hundreds of samples ofmore » small proteins. For example, the previously published mechanism of 53 kDa cholesteryl ester transfer protein (CETP) involved the screening and imaging of hundreds of samples. Considering cryo-EM rarely successfully images proteins less than 200 kDa has yet to publish any study involving screening over one hundred sample conditions, it is fair to call OpNS a high-throughput method for studying small proteins. Hopefully the OpNS protocol presented here can be a useful tool to push the boundaries of EM and accelerate EM studies into small protein structure, dynamics and mechanisms.« less

Authors:
 [1];  [1];  [1]
  1. Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States). The Molecular Foundry
Publication Date:
Research Org.:
Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States)
Sponsoring Org.:
USDOE Office of Science (SC), Basic Energy Sciences (BES) (SC-22); National Inst. of Health (NIH) (United States)
OSTI Identifier:
1407250
Grant/Contract Number:  
AC02-05CH11231; R01HL115153; R01GM104427
Resource Type:
Journal Article: Accepted Manuscript
Journal Name:
Journal of Visualized Experiments
Additional Journal Information:
Journal Volume: 90; Journal ID: ISSN 1940-087X
Publisher:
MyJoVE Corp.
Country of Publication:
United States
Language:
English
Subject:
47 OTHER INSTRUMENTATION; small and asymmetric protein structure; electron microscopy; optimized negative staining

Citation Formats

Rames, Matthew, Yu, Yadong, and Ren, Gang. Optimized Negative Staining: a High-throughput Protocol for Examining Small and Asymmetric Protein Structure by Electron Microscopy. United States: N. p., 2014. Web. doi:10.3791/51087.
Rames, Matthew, Yu, Yadong, & Ren, Gang. Optimized Negative Staining: a High-throughput Protocol for Examining Small and Asymmetric Protein Structure by Electron Microscopy. United States. doi:10.3791/51087.
Rames, Matthew, Yu, Yadong, and Ren, Gang. Fri . "Optimized Negative Staining: a High-throughput Protocol for Examining Small and Asymmetric Protein Structure by Electron Microscopy". United States. doi:10.3791/51087. https://www.osti.gov/servlets/purl/1407250.
@article{osti_1407250,
title = {Optimized Negative Staining: a High-throughput Protocol for Examining Small and Asymmetric Protein Structure by Electron Microscopy},
author = {Rames, Matthew and Yu, Yadong and Ren, Gang},
abstractNote = {Structural determination of proteins is rather challenging for proteins with molecular masses between 40 - 200 kDa. Considering that more than half of natural proteins have a molecular mass between 40 - 200 kDa, a robust and high-throughput method with a nanometer resolution capability is needed. Negative staining (NS) electron microscopy (EM) is an easy, rapid, and qualitative approach which has frequently been used in research laboratories to examine protein structure and protein-protein interactions. Unfortunately, conventional NS protocols often generate structural artifacts on proteins, especially with lipoproteins that usually form presenting rouleaux artifacts. By using images of lipoproteins from cryo-electron microscopy (cryo-EM) as a standard, the key parameters in NS specimen preparation conditions were recently screened and reported as the optimized NS protocol (OpNS), a modified conventional NS protocol. Artifacts like rouleaux can be greatly limited by OpNS, additionally providing high contrast along with reasonably high-resolution (near 1 nm) images of small and asymmetric proteins. These high-resolution and high contrast images are even favorable for an individual protein (a single object, no average) 3D reconstruction, such as a 160 kDa antibody, through the method of electron tomography. Moreover, OpNS can be a high-throughput tool to examine hundreds of samples of small proteins. For example, the previously published mechanism of 53 kDa cholesteryl ester transfer protein (CETP) involved the screening and imaging of hundreds of samples. Considering cryo-EM rarely successfully images proteins less than 200 kDa has yet to publish any study involving screening over one hundred sample conditions, it is fair to call OpNS a high-throughput method for studying small proteins. Hopefully the OpNS protocol presented here can be a useful tool to push the boundaries of EM and accelerate EM studies into small protein structure, dynamics and mechanisms.},
doi = {10.3791/51087},
journal = {Journal of Visualized Experiments},
issn = {1940-087X},
number = ,
volume = 90,
place = {United States},
year = {2014},
month = {8}
}

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