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Fast Small-Scale Membrane Protein Purification and Grid Preparation for Single-Particle Electron Microscopy

  • Natalie Bärland
  • Camilo PerezEmail author
Protocol
  • 496 Downloads
Part of the Methods in Molecular Biology book series (MIMB, volume 2127)

Abstract

The ongoing development of single-particle cryo-electron microscopy (cryo-EM) is leading to fast data acquisition, data processing, and protein structure elucidation. Quick and reliable methods to go from protein purification and optimization to grid preparation will significantly improve the reach and power of cryo-EM. Such methods would particularly constitute a tremendous advantage in structural biology of membrane proteins, whose published structures stay still far behind the number of soluble protein structures. Here we describe a fast, low-cost, and user-friendly method for the purification and cryo-EM analysis of a recombinant membrane protein. This method minimizes the amount of starting material and manipulation steps needed to go from purification to grid preparation, and could potentially be expanded to other membrane protein purification systems for its direct application in structure determination by single-particle cryo-EM.

Key words

Membrane proteins Affinity-chromatography Small-scale purification Single-particle electron microscopy Negative staining-EM Cryo-EM 

Notes

Acknowledgments

We thank the staff at the BioEM Lab and Nano Imaging Lab of the University of Basel. This work was supported by the Swiss National Science Foundation (SNSF) (PP00P3_170607).

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Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2020

Authors and Affiliations

  1. 1.BiozentrumUniversity of BaselBaselSwitzerland

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