Qubit protein assay kit with qubit 2.0 fluorometer

Protein concentration of the exosome fraction was determined using a Qubit Protein Assay Kit with Qubit 2.0 Fluorometer (Invitrogen, Carlsbad, CA, USA). Exosome fractions (1 μg protein) were separated using 10% Novex Bis‐Tris Protein Gels (Invitrogen), transferred to PVDF membranes (Invitrogen), and immunoblotted with the following primary antibodies: CD9 (1:200; Santa Cruz Biotechnology, Dallas, TX, USA) and CD63 (1:1000; Medical & Biological Laboratories, Nagoya, Japan). Antimouse immunoglobulin (Ig)G, peroxidase‐linked antibody (GE Healthcare, Buckinghamshire, UK) was used as secondary antibody. Chemiluminescence was detected using Amersham ECL Prime Western Blotting Detection Reagents (GE Healthcare) and imaged using LAS‐3000 mini (Fujifilm Life Science, Tokyo, Japan).

Protein concentration of exosome fraction was determined using a Qubit Protein Assay Kit with Qubit 2.0 Fluorometer (Invitrogen; Thermo Fisher Scientific, Inc.). The exosome fractions (1 µg of protein) were directly mixed with loading buffer and heated at 70°C for 10 min as previously described (21 (link)) and loaded and separated using 10% Novex Bis-Tris Protein Gels (Invitrogen; Thermo Fisher Scientific, Inc.), and then transferred onto PVDF membranes (Invitrogen), and immunoblotted with the following primary antibodies (incubated overnight at 4°C): CD9 (dilution 1:200; cat. no. sc-59140; Santa Cruz Biotechnology, Inc.) and CD63 (dilution 1:1,000; cat. no. MEX002-3; Medical & Biological Laboratories). Anti-mouse IgG, peroxidase-linked antibody (dilution 1:50,000; cat. no. NA931; GE Healthcare) was used as a secondary antibody (incubated for 1 h at room temprature). Chemiluminescence was detected using Amersham ECL Prime Western Blotting Detection Reagents (GE Healthcare Bio-Sciences) and imaged using LAS-3000 mini (Fujifilm Life Sciences).