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. 1991 Mar 2;112(6):1133–1141. doi: 10.1083/jcb.112.6.1133

Isolation of functional, coated, endocytic vesicles

PMCID: PMC2288903  PMID: 1900301

Abstract

Brief internalization of [125I]transferrin was used to label coated endocytic vesicles, which were then purified using a combination of 2H2O and 2H2O/Ficoll density gradients. Purification was monitored using an assay measuring fusion of endocytic organelles, so as to isolate functional vesicles. Isolated vesicles had all the properties of clathrin-coated vesicles, being enriched for the major components of clathrin coats and uncoated by either 1 M Tris-HCl or an uncoating ATPase. Nearly half of the labeled vesicles were able to participate in subsequent fusion events, as measured by the cell-free assay. Fusion was specific, requiring energy and cytosol, and being sensitive to N- ethyl maleimide.

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Selected References

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