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. 2021:2215:115-123.
doi: 10.1007/978-1-0716-0966-8_5.

Progress Towards CryoEM: Negative-Stain Procedures for Biological Samples

Shane Gonen  1
Affiliations
Free article

Progress Towards CryoEM: Negative-Stain Procedures for Biological Samples

Shane Gonen. Methods Mol Biol. 2021.
Free article

Abstract

In recent years, electron cryo-microscopy (CryoEM) has become a powerful method for the high-resolution studies of biological macromolecules. While CryoEM experiments can begin without additional microscopy steps, negative-stain EM can tremendously minimize CryoEM screening. Negative-stain is a quick method that can be used to screen for robust biochemical conditions, the integrity, binding, and composition of samples and to get an estimation of sample grid concentration. For some applications, the map resolutions potentially afforded by stain may be as biologically informative as in CryoEM. Here, I describe the benefits and pitfalls of negative-stain EM, with particular emphasis on Uranyl stains with the main goal of screening in advance of CryoEM. In addition, I provide a materials list, detailed protocol and possible adjustments for the use of stains for biological samples requiring imaging and/or diffraction-based methods of EM.

Keywords: Averaging; CryoEM; Crystals; Electron crystallography; Electron microscopy; Negative-stain; Single-particle; Uranyl acetate; Uranyl formate.

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