Quantifying Extracellular Vesicle Proteins

The concentration of total protein in EV samples and cells lysed in RIPA buffer was determined using the NanoOrange^™ protein quantitation kit (#N6666, ThermoFisher Scientific, Eugene, OR, USA) according to the manufacturer’s recommendations using a SpectraMax M5e microplate reader (Molecular Devices, LLC., San Jose, CA, USA). Immunoblotting was performed according to the previously described procedure [25 (link)] with the differences that 5 µg of total protein was applied to SDS-PAGE and proteins were visualized with SuperSignal^™ West Femto Maximum Sensitivity Substrate (#34095, ThermoFisher Scientific, Rockford, IL, USA). The following primary and secondary antibodies and dilutions were used: anti-Alix (#sc-271975, 1:500; Santa Cruz Biotechnology, Dallas, TX, USA), anti-Flotillin-2 (#3436S, 1:1000; Cell Signaling Technology, Topsfield, MA, USA), anti-CD9 (#13174, 1:2000; Cell Signaling Technology), anti-TSG-101 (ab125011, 1:5000; Abcam, Cambridge, UK), anti-PCNA (#sc-7907, 1:500; Santa Cruz Biotechnology), anti-Stomatin (#sc-134554, 1:500; Santa Cruz Biotechnology), anti-mouse goat polyclonal antibodies (#2367, 1:5000; Cell Signaling Technology); and anti-rabbit goat polyclonal antibodies (#29902, 1:80,000; Cell Signaling Technology).