Extracellular Vesicles pp 143-152

Part of the Methods in Molecular Biology book series (MIMB, volume 1660) | Cite as

Extracellular Vesicle Isolation and Analysis by Western Blotting

  • Emma J. K. Kowal
  • Dmitry Ter-Ovanesyan
  • Aviv Regev
  • George M. Church
Protocol

Abstract

Extracellular vesicles (EVs) are released by mammalian cells and are thought to be important mediators of intercellular communication. There are many methods for isolating EVs from cell culture media, but the most popular methods involve purification based on ultracentrifugation. Here, we provide a detailed protocol for isolating EVs by differential ultracentrifugation and analyzing EV proteins (such as the tetraspaninsCD9, CD63 and CD81) by western blotting.

Key words

Exosomes Extracellular vesicles Exosome isolation Extracellular vesicle isolation Ultracentrifugation Immunoblotting Western blot Tetraspanins CD63 CD81 CD9 

References

  1. 1.
    Tkach M, Thery C (2016) Communication by extracellular vesicles: where we are and where we need to go. Cell 164(6):1226–1232CrossRefPubMedGoogle Scholar
  2. 2.
    Gould SJ, Raposo G (2013) As we wait: coping with an imperfect nomenclature for extracellular vesicles. J Extracell Vesicles 2. doi:10.3402/jev.v2i0.20389
  3. 3.
    Witwer KW, Buzas EI, Bemis LT, Bora A, Lasser C, Lotvall J, Nolte-‘t Hoen EN, Piper MG, Sivaraman S, Skog J, Thery C, Wauben MH, Hochberg F (2013) Standardization of sample collection, isolation and analysis methods in extracellular vesicle research. J Extracell Vesicles 2. doi:10.3402/jev.v2i0.20360
  4. 4.
    Thery C, Amigorena S, Raposo G, Clayton A (2006) Isolation and characterization of exosomes from cell culture supernatants and biological fluids. Curr Protoc Cell Biol Chapter 3:Unit 3.22Google Scholar
  5. 5.
    Kowal J, Arras G, Colombo M, Jouve M, Morath JP, Primdal-Bengtson B, Dingli F, Loew D, Tkach M, Thery C (2016) Proteomic comparison defines novel markers to characterize heterogeneous populations of extracellular vesicle subtypes. Proc Natl Acad Sci U S A 113(8):E968–E977CrossRefPubMedPubMedCentralGoogle Scholar
  6. 6.
    XCell SureLock ® Mini-Cell. https://tools.thermofisher.com/content/sfs/manuals/surelock_man.pdf. Accessed 27 Apr 2016
  7. 7.
    Ageberg M, Lindmark A (2003) Characterisation of the biosynthesis and processing of the neutrophil granule membrane protein CD63 in myeloid cells. Clin Lab Haematol 25(5):297–306CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media LLC 2017

Authors and Affiliations

  • Emma J. K. Kowal
    • 1
    • 2
  • Dmitry Ter-Ovanesyan
    • 1
    • 2
    • 3
    • 4
  • Aviv Regev
    • 4
    • 5
  • George M. Church
    • 1
    • 2
  1. 1.Department of GeneticsHarvard Medical SchoolBostonUSA
  2. 2.Wyss Institute for Biologically Inspired EngineeringHarvard UniversityBostonUSA
  3. 3.Department of Molecular and Cellular BiologyHarvard UniversityCambridgeUSA
  4. 4.Broad Institute of MIT and HarvardCambridgeUSA
  5. 5.Department of BiologyMITCambridgeUSA

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