Preparation of Enriched Plant Clathrin-Coated Vesicles by Differential and Density Gradient Centrifugation
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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 wordsClathrin Fractionation Negative stain electron microscopy Plant Vesicles
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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