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Preparation of Enriched Plant Clathrin-Coated Vesicles by Differential and Density Gradient Centrifugation

  • Gregory D. Reynolds
  • Ben August
  • Sebastian Y. BednarekEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1209)

Abstract

Methods for the subcellular fractionation and enrichment of specific intracellular compartments are essential tools in the analysis of compartment composition and function. In vitro characterization of isolated cell organelles and other endomembrane intermediates, including exploration of the compartment protein ensemble, offers strong clues of in vivo function identity. Here, we describe methodology for the isolation of clathrin-coated vesicles from Arabidopsis thaliana suspension-cultured cells on the basis of differential and density centrifugation.

Key words

Clathrin Fractionation Negative stain electron microscopy Plant Vesicles 

Notes

Acknowledgments

This work was supported by funding provided by the National Science Foundation (No. 1121998) and a Vilas Associate Award (University of Wisconsin, Madison, Graduate School) to S.Y.B. The authors would also like to thank Nou Vang for technical assistance in the preparation and quantitative analysis of CCVs.

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Copyright information

© Springer Science+Business Media New York 2014

Authors and Affiliations

  • Gregory D. Reynolds
    • 1
  • Ben August
    • 2
  • Sebastian Y. Bednarek
    • 1
    Email author
  1. 1.Department of BiochemistryUniversity of Wisconsin-MadisonMadisonUSA
  2. 2.Electron Microscope Facility, School of Public HealthUniversity of Wisconsin-MadisonMadisonUSA

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