Protocol

Membrane Proteomics

Volume 528 of the series Methods in Molecular Biology™ pp 227-242

Isolation of Extracellular Membranous Vesicles for Proteomic Analysis

  • Rommel A. MathiasAffiliated withJoint Proteomics Laboratory, Ludwig Institute for Cancer Research & The Walter and Eliza Hall Institute of Medical Research
  • , Justin W. LimAffiliated withDepartment of Biochemistry and Molecular Biology, University of Melbourne
  • , Hong JiAffiliated withJoint Proteomics Laboratory, Ludwig Institute for Cancer Research & The Walter and Eliza Hall Institute of Medical Research
  • , Richard J. SimpsonAffiliated withJoint ProteomicS Laboratory, Ludwig Institute for Cancer Research & The Walter and Eliza Hall Institute of Medical Research, Royal Melbourne Hospital

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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