Isolation of extracellular membranous vesicles for proteomic analysis.

Rommel A. Mathias, Justin W. Lim, Hong Ji, Richard J. Simpson

Research output: Contribution to journalArticleResearchpeer-review

36 Citations (Scopus)

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.

Original languageEnglish
Pages (from-to)227-242
Number of pages16
JournalMethods in Molecular Biology
Volume528
DOIs
Publication statusPublished - 2009
Externally publishedYes

Cite this