Skip to main content
SpringerLink
Log in
Book cover

Exocytosis and Endocytosis pp 85–99Cite as

Fractionation of Subcellular Membrane Vesicles of Epithelial and Non-epithelial Cells by OptiPrep™ Density Gradient Ultracentrifugation

  • Protocol
  • First Online:

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1174))

Abstract

Density gradient ultracentrifugation (DGUC) is widely used for physical isolation (enrichment rather than purification) of subcellular membrane vesicles. It has been a valuable tool to study specific subcellular localization and dynamic trafficking of proteins. While sucrose has been the main component of density gradients, several years ago, synthetic OptiPrep™ (iodixanol) began being used for separation of organelles due to its iso-osmotic property. Here, we describe a detailed protocol for density gradient fractionation of various mammalian subcellular vesicles, including endoplasmic reticulum (ER), Golgi apparatus, endosomes, and lipid rafts, as well as apical and basolateral membranes of polarized epithelial cells.

This is a preview of subscription content, log in via an institution.

Protocol
USD   49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD   89.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD   119.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD   169.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Learn about institutional subscriptions

Springer Nature is developing a new tool to find and evaluate Protocols. Learn more

References

  1. Ngsee JK, Trimble WS, Elferink LA et al (1990) Molecular analysis of proteins associated with the synaptic vesicle membrane. Cold Spring Harb Symp Quant Biol 55:111–118

    Article  CAS  PubMed  Google Scholar 

  2. Li X, Sze H (1999) A 100 kDa polypeptide associates with the V0 membrane sector but not with the active oat vacuolar H(+)-ATPase, suggesting a role in assembly. Plant J 17:19–30

    Article  PubMed  Google Scholar 

  3. Huber LA, Pfaller K, Vietor I (2003) Organelle proteomics: implications for subcellular fractionation in proteomics. Circ Res 92:962–968

    Article  CAS  PubMed  Google Scholar 

  4. Gutwein P, Stoeck A, Riedle S et al (2005) Cleavage of L1 in exosomes and apoptotic membrane vesicles released from ovarian carcinoma cells. Clin Cancer Res 11:2492–2501

    Article  CAS  PubMed  Google Scholar 

  5. Ford T, Graham J, Rickwood D (1994) Iodixanol: a nonionic iso-osmotic centrifugation medium for the formation of self-generated gradients. Anal Biochem 220:360–366

    Article  CAS  PubMed  Google Scholar 

  6. Graham J, Ford T, Rickwood D (1994) The preparation of subcellular organelles from mouse liver in self-generated gradients of iodixanol. Anal Biochem 220:367–373

    Article  CAS  PubMed  Google Scholar 

  7. Li X, Galli T, Leu S et al (2001) Na + -H + exchanger 3 (NHE3) is present in lipid rafts in the rabbit ileal brush border: a role for rafts in trafficking and rapid stimulation of NHE3. J Physiol 537:537–552

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  8. Li X, Leu S, Cheong A et al (2004) Akt2, phosphatidylinositol 3-kinase, and PTEN are in lipid rafts of intestinal cells: role in absorption and differentiation. Gastroenterology 126:122–135

    Article  CAS  PubMed  Google Scholar 

  9. Janecki AJ, Montrose MH, Tse CM, de Medina FS, Zweibaum A, Donowitz M (1999) Development of an endogenous epithelial Na(+)/H(+) exchanger (NHE3) in three clones of caco-2 cells. Am J Physiol 277:G292–G305

    CAS  PubMed  Google Scholar 

  10. Cohen ME, Wesolek J, McCullen J et al (1991) Carbachol- and elevated Ca(2+)-induced translocation of functionally active protein kinase C to the brush border of rabbit ileal Na + absorbing cells. J Clin Invest 88:855–863

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  11. Simons K, Toomre D (2000) Lipid rafts and signal transduction. Nat Rev Mol Cell Biol 1:31–39

    Article  CAS  PubMed  Google Scholar 

  12. Simons K, Vaz WL (2004) Model systems, lipid rafts, and cell membranes. Annu Rev Biophys Biomol Struct 33:269–295

    Article  CAS  PubMed  Google Scholar 

  13. Brown DA (2006) Lipid rafts, detergent-resistant membranes, and raft targeting signals. Physiology (Bethesda) 21:430–439

    Article  CAS  Google Scholar 

  14. Lusa S, Blom TS, Eskelinen EL et al (2001) Depletion of rafts in late endocytic membranes is controlled by NPC1- dependent recycling of cholesterol to the plasma membrane. J Cell Sci 114:1893–1900

    CAS  PubMed  Google Scholar 

  15. Lafont F, Verkade P, Galli T, Wimmer C, Louvard D, Simons K (1999) Raft association of SNAP receptors acting in apical trafficking in Madin-Darby canine kidney cells. Proc Natl Acad Sci U S A 96:3734–3738

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  16. Bagnat M, Keranen S, Shevchenko A, Shevchenko A, Simons K (2000) Lipid rafts function in biosynthetic delivery of proteins to the cell surface in yeast. Proc Natl Acad Sci U S A 97:3254–3259

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  17. Fullekrug J, Simons K (2004) Lipid rafts and apical membrane traffic. Ann N Y Acad Sci 1014:164–169

    Article  PubMed  Google Scholar 

Download references

Acknowledgments

This work is supported by in part by the National Institutes of Health, NIDDK Grants KO1-DK62264, RO1-DK26523, RO1-DK61765, PO1-DK44484, PO1-DK72084, R24-DK64388, Broad Medical Research Program Grant (IBD-0119R2), and the Hopkins Center for Epithelial Disorders. We thank the Elsevier Inc. for permission to reuse the figures originally published in its journal Gastroenterology.

Author information

Authors and Affiliations

  1. GI Division, Department of Medicine, Johns Hopkins University School of Medicine, 918 Ross Research Bldg, 720 Rutland Ave, 21205, Baltimore, MD, USA

    Xuhang Li & Mark Donowitz

Authors
  1. Xuhang Li
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Mark Donowitz
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Xuhang Li .

Editor information

Editors and Affiliations

  1. Department of Human and Molecular Genetics, VCU Institute of Molecular Medicine, Virginia Commonwealth University, Richmond, Virginia, USA

    Andrei I. Ivanov

Rights and permissions

Reprints and permissions

Copyright information

© 2014 Springer Science+Business Media New York

About this protocol

Cite this protocol

Li, X., Donowitz, M. (2014). Fractionation of Subcellular Membrane Vesicles of Epithelial and Non-epithelial Cells by OptiPrep™ Density Gradient Ultracentrifugation. In: Ivanov, A. (eds) Exocytosis and Endocytosis. Methods in Molecular Biology, vol 1174. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-0944-5_6

Download citation

  • DOI: https://doi.org/10.1007/978-1-4939-0944-5_6

  • Published:

  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-0943-8

  • Online ISBN: 978-1-4939-0944-5

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation