Isolation of extracellular membranous vesicles for proteomic analysis.

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

doi:10.1007/978-1-60327-310-7_16

Isolation of extracellular membranous vesicles for proteomic analysis.

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

Research output: Contribution to journal › Article › Research › peer-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 language	English
Pages (from-to)	227-242
Number of pages	16
Journal	Methods in Molecular Biology
Volume	528
DOIs	https://doi.org/10.1007/978-1-60327-310-7_16
Publication status	Published - 2009
Externally published	Yes

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10.1007/978-1-60327-310-7_16

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Cite this

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title = "Isolation of extracellular membranous vesicles for proteomic analysis.",

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.",

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AB - 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.

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