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Preparation of Disease-Related Protein Assemblies for Single Particle Electron Microscopy

  • A. Cameron Varano
  • Naoe Harafuji
  • William Dearnaley
  • Lisa Guay-Woodford
  • Deborah F. Kelly
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1647)

Abstract

Electron microscopy (EM) is a rapidly growing area of structural biology that permits us to decode biological assemblies at the nanoscale. To examine biological materials for single particle EM analysis, purified assemblies must be obtained using biochemical separation techniques. Here, we describe effective methodologies for isolating histidine (his)-tagged protein assemblies from the nucleus of disease-relevant cell lines. We further demonstrate how isolated assemblies are visualized using single particle EM techniques and provide representative results for each step in the process.

Key words

Recombinant proteins Histidine tag Fibrocystin Electron microscopy Single particle analysis 

Notes

Acknowledgment

This work was supported by NIH/NCI grant R01CA193578 to D.F.K.

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Copyright information

© Springer Science+Business Media LLC 2017

Authors and Affiliations

  • A. Cameron Varano
    • 1
    • 2
  • Naoe Harafuji
    • 3
    • 4
  • William Dearnaley
    • 1
  • Lisa Guay-Woodford
    • 3
    • 4
  • Deborah F. Kelly
    • 1
    • 5
  1. 1.Virginia Tech Carilion Research InstituteRoanokeUSA
  2. 2.Translational Biology, Medicine and Health Graduate ProgramVirginia TechBlacksburgUSA
  3. 3.Center for Translational Science, Children’s National Health SystemWashington, DCUSA
  4. 4.Department of PediatricsGeorge Washington University School of Medicine and Health SciencesWashington, DCUSA
  5. 5.Department of Biological SciencesVirginia TechBlacksburgUSA

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