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Immunogold Electron Microscopy

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Updated: May 18, 2026

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

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

Published on: August 15, 2014

Optimized negative-staining electron microscopy for lipoprotein studies.

Lei Zhang¹, Huimin Tong, Mark Garewal

¹The Molecular Foundry, Lawrence Berkeley National Laboratory, Berkeley, CA, USA.

Biochimica Et Biophysica Acta

|October 4, 2012

Summary

This summary is machine-generated.

An optimized negative-staining (OpNS) protocol effectively removes artifacts in electron microscopy (EM) imaging of lipoproteins. This advanced OpNS method provides high-resolution structural insights for dynamic proteins.

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

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

Published on: August 15, 2014

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Published on: February 6, 2018

Area of Science:

Structural Biology
Biophysics
Electron Microscopy

Background:

Negative-staining (NS) electron microscopy (EM) is a long-standing technique for initial protein structure studies.
Conventional NS protocols can introduce artifacts, particularly for flexible or lipid-rich proteins like lipoproteins, leading to rouleau formation.
Lipoprotein structure is sensitive to NS sample preparation variables, including salt concentration and staining reagents.

Purpose of the Study:

To review popular negative-staining protocols for lipoprotein morphology and structure analysis.
To evaluate an optimized negative-staining (OpNS) protocol for its efficacy in mitigating artifacts and characterizing lipoprotein structures.

Main Methods:

Review of established negative-staining (NS) electron microscopy (EM) protocols.
Application and comparison of an optimized NS (OpNS) protocol with conventional methods and cryo-electron microscopy (cryo-EM).

Main Results:

The optimized NS (OpNS) protocol successfully eliminated rouleau artifacts observed with conventional NS.
Lipoprotein size and shape determined by OpNS align with cryo-electron microscopy (cryo-EM) measurements.
OpNS achieved high throughput, contrast, and resolution (near 1nm), enabling studies like cholesterol ester transfer protein (CETP) mechanics and IgG antibody 3D structure.

Conclusions:

OpNS is a superior method for studying lipoprotein structure, avoiding common artifacts.
OpNS offers a reliable, high-resolution approach for characterizing protein structures, including dynamic and flexible proteins.
The optimized protocol is recommended for general application in protein structure determination, especially for dynamic proteins.