Isolation of clathrin-coated vesicles by differential and density gradient centrifugation
- PMID: 18228473
- DOI: 10.1002/0471143030.cb0313s26
Isolation of clathrin-coated vesicles by differential and density gradient centrifugation
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 D(2)O-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.
Similar articles
-
Purification of Clathrin-Coated Vesicles from Adult Rat Brain.Methods Mol Biol. 2018;1847:1-11. doi: 10.1007/978-1-4939-8719-1_1. Methods Mol Biol. 2018. PMID: 30129005
-
Preparation of enriched plant clathrin-coated vesicles by differential and density gradient centrifugation.Methods Mol Biol. 2014;1209:163-77. doi: 10.1007/978-1-4939-1420-3_13. Methods Mol Biol. 2014. PMID: 25117283
-
Isolation of Golgi membranes from tissues and cells by differential and density gradient centrifugation.Curr Protoc Cell Biol. 2001 May;Chapter 3:Unit 3.9. doi: 10.1002/0471143030.cb0309s10. Curr Protoc Cell Biol. 2001. PMID: 18228361
-
Proteomic analysis of clathrin-coated vesicles.Proteomics. 2010 Nov;10(22):4025-39. doi: 10.1002/pmic.201000253. Proteomics. 2010. PMID: 21080493 Review.
-
Isolation of endocitic organelles by density gradient centrifugation.Methods Mol Biol. 2008;424:317-31. doi: 10.1007/978-1-60327-064-9_25. Methods Mol Biol. 2008. PMID: 18369872 Review.
Cited by
-
The Connecdenn DENN domain: a GEF for Rab35 mediating cargo-specific exit from early endosomes.Mol Cell. 2010 Feb 12;37(3):370-82. doi: 10.1016/j.molcel.2009.12.037. Mol Cell. 2010. PMID: 20159556 Free PMC article.
-
ORF33 and ORF38 of Kaposi's Sarcoma-Associated Herpesvirus Interact and Are Required for Optimal Production of Infectious Progeny Viruses.J Virol. 2015 Dec 4;90(4):1741-56. doi: 10.1128/JVI.02738-15. Print 2016 Feb 15. J Virol. 2015. PMID: 26637455 Free PMC article.
-
NECAP 1 regulates AP-2 interactions to control vesicle size, number, and cargo during clathrin-mediated endocytosis.PLoS Biol. 2013 Oct;11(10):e1001670. doi: 10.1371/journal.pbio.1001670. Epub 2013 Oct 1. PLoS Biol. 2013. PMID: 24130457 Free PMC article.
-
The Mammalian Ribo-interactome Reveals Ribosome Functional Diversity and Heterogeneity.Cell. 2017 Jun 1;169(6):1051-1065.e18. doi: 10.1016/j.cell.2017.05.022. Cell. 2017. PMID: 28575669 Free PMC article.
-
The connecdenn family, Rab35 guanine nucleotide exchange factors interfacing with the clathrin machinery.J Biol Chem. 2010 Apr 2;285(14):10627-37. doi: 10.1074/jbc.M109.050930. Epub 2010 Feb 12. J Biol Chem. 2010. PMID: 20154091 Free PMC article.
MeSH terms
LinkOut - more resources
Full Text Sources
