Vesicle Protein Content and Size Analysis

Vesicle protein content was determined by BCA (Bio-Rad). The particle concentration and size distribution of each fraction collected after (i) ultracentrifugation only and (ii) iodixanol gradient was determined by TRPS using a qNano instrument (Izon Science, New Zealand) following the manufacturer’s instructions. Briefly, a nanopore NP150 was used, and pressure and voltage values were set to optimize the signal to ensure high sensitivity. Samples were diluted 1:5 in electrolyte (Izon Science) before applying to the nanopore. Calibration was performed using CP100 carboxylated polystyrene calibration particles (Izon Science) at a 1:2,000 dilution. Size distribution and concentration of particles were analyzed using the software provided by Izon (version 3.2), and purity analysis (number of particles/µg of protein) was performed following established methods (Webber and Clayton, 2013 (link)).

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Chaiyadet S., Sotillo J., Krueajampa W., Thongsen S., Smout M., Brindley P.J., Laha T, & Loukas A. (2022). Silencing of Opisthorchis viverrini Tetraspanin Gene Expression Results in Reduced Secretion of Extracellular Vesicles. Frontiers in Cellular and Infection Microbiology, 12, 827521.

Publication 2022

BCA qNano CP100 carboxylated polystyrene calibration particles

Dilution Electrolyte Iodixanol Polystyrene Pressure Protein Sensitivity Trps Ultracentrifugation

Corresponding Organization : James Cook University

Other organizations : Instituto de Salud Carlos III, George Washington University

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Variable analysis

independent variables

Ultracentrifugation only
Iodixanol gradient

dependent variables

Particle concentration
Size distribution of particles
Protein content of vesicles

control variables

Nanopore NP150 used for TRPS
Pressure and voltage values optimized for high sensitivity
Samples diluted 1:5 in electrolyte before applying to nanopore
Calibration performed using CP100 carboxylated polystyrene calibration particles at 1:2,000 dilution

controls

Positive control: CP100 carboxylated polystyrene calibration particles
Negative control: Not explicitly mentioned

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