Type

Journal Article

Authors

Harry Holthofer
Paula Meleady
Michael Henry
Dontscho Kerjaschki
Dorota Tataruch-Weinert
Luca Musante

Subjects

Chemistry

Topics
dialysis size mass spectrometry transmission electron microscopy tem filtration characterisation western blot ultracentrifugation

Residual urinary extracellular vesicles in ultracentrifugation supernatants after hydrostatic filtration dialysis enrichment. (2016)

Abstract Urinary extracellular vesicles (UEVs) appear an ideal source of biomarkers for kidney and urogenital diseases. The majority of protocols designed for their isolation are based on differential centrifugation steps. However, little is still known of the type and amount of vesicles left in the supernatant. Here we used an isolation protocol for UEVs which uses hydrostatic filtration dialysis as first pre-enrichment step, followed by differential centrifugation. Transmission electron microscopy (TEM), mass spectrometry (MS), western blot, ELISA assays and tuneable resistive pulse sensing (TRPS) were used to characterise and quantify UEVs in the ultracentrifugation supernatant. TEM showed the presence of a variety of small size vesicles in the supernatant while protein identification by MS matched accurately with the protein list available in Vesiclepedia. Screening and relative quantification for specific vesicle markers showed that the supernatant was preferentially positive for CD9 and TSG101. ELISA tests for quantification of exosome revealed that 14%, was left in the supernatant with a particle diameter of 110 nm and concentration of 1.54 × 10(10)/ml. Here we show a comprehensive characterisation of exosomes and other small size urinary vesicles which the conventional differential centrifugation protocol may lose.
Collections Ireland -> Dublin City University -> PubMed

Full list of authors on original publication

Harry Holthofer, Paula Meleady, Michael Henry, Dontscho Kerjaschki, Dorota Tataruch-Weinert, Luca Musante

Experts in our system

1
Harry Holthofer
Dublin City University
Total Publications: 18
 
2
Paula Meleady
Dublin City University
Total Publications: 115
 
3
Michael Henry
Dublin City University
Total Publications: 99
 
4
Luca Musante
Dublin City University
Total Publications: 14