Unit

UNIT 3.13 Isolation of Clathrin-Coated Vesicles by Differential and Density Gradient Centrifugation

  1. Martine Girard,
  2. Patrick D. Allaire,
  3. Francois Blondeau,
  4. Peter S. McPherson

Published Online: 1 APR 2005

DOI: 10.1002/0471143030.cb0313s26

Current Protocols in Cell Biology

Current Protocols in Cell Biology

How to Cite

Girard, M., Allaire, P. D., Blondeau, F. and McPherson, P. S. 2005. Isolation of Clathrin-Coated Vesicles by Differential and Density Gradient Centrifugation. Current Protocols in Cell Biology. 26:3.13:3.13.1–3.13.31.

Author Information

  1. Montreal Neurological Institute, McGill University, Montreal, Quebec, Canada

Publication History

  1. Published Online: 1 APR 2005
  2. Published Print: MAR 2005

Abstract

Clathrin-coated vesicles (CCVs) are an important class of transport organelles that mediate the endocytosis of proteins and lipids at the plasma membrane and the transport of proteins from the trans-Golgi network to the endosomal/lysosomal system. The authors describe a protocol for isolating CCVs from adult rat brain using differential centrifugation, Ficoll and D2O-sucrose density gradient centrifugation, and velocity sedimentation in linear sucrose gradients. The application of this basic method to the isolation of CCVs from developing rat brains and to the generation of relatively crude CCVs from cultured cells is also described. Furthermore, we describe a protocol in which differential centrifugation and a series of discontinuous sucrose gradients are used to isolate CCVs from rat liver. An approach to analyzing CCV purity by electron microscopy is also described.