Isolation of Extracellular Membranous Vesicles for Proteomic Analysis

  • Rommel A. Mathias
  • Justin W. Lim
  • Hong Ji
  • Richard J. Simpson
Part of the Methods in Molecular Biology™ book series (MIMB, volume 528)

Abstract

Membranous vesicles are constitutively released by a multitude of cell types. Following fusion of multivesicular bodies with the plasma membrane, endocytic vesicles, 30–90 nm in size termed exosomes are released extracellularly. Whilst several groups have reported the presence of exosomes in cell-culture conditioned medium, their biological and physiological functions still remain unclear. In addition, exosomes have been detected in body fluids associated with disease, further demonstrating their potential as diagnostic biomarkers. This protocol employs size filtration followed by ultracentrifugation to isolate and purify exosomes from the colon carcinoma cell line LIM 1215. Morphological visualisation and characterisation is based on electron microscopy and western blotting, whilst protein identification is achieved using a combination of 1D SDS-PAGE and LC-MS/MS.

Key words

Membranous vesicles conditioned medium exosomes LIM 1215 proteomics 

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Copyright information

© Humana Press, a part of Springer Science+Business Media, LLC 2009

Authors and Affiliations

  • Rommel A. Mathias
    • 1
  • Justin W. Lim
    • 2
  • Hong Ji
    • 3
  • Richard J. Simpson
    • 4
  1. 1.Joint Proteomics LaboratoryLudwig Institute for Cancer Research & The Walter and Eliza Hall Institute of Medical ResearchMelbourneAustralia
  2. 2.Department of Biochemistry and Molecular BiologyUniversity of MelbourneMelbourneAustralia
  3. 3.Joint Proteomics LaboratoryLudwig Institute for Cancer Research & The Walter and Eliza Hall Institute of Medical ResearchMelbourneAustralia
  4. 4.Joint ProteomicS LaboratoryLudwig Institute for Cancer Research & The Walter and Eliza Hall Institute of Medical Research, Royal Melbourne HospitalParkvilleAustralia

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