Quantifying Exosomal Proteins by Western Blot

Protein-based quantitation of isolated exosomes was done using the protein DC assay kit as described earlier by us^{7 (link),34 (link)}. Subsequently, equal amount of exosomes (25 μg) collected from different isolation methods was denatured using 6X denaturation buffer at 95 °C for 10 min and then resolved on 12% SDS-Polyacrylamide gel by electrophoresis. Resolved proteins were transferred onto Immobilon-P PVDF membrane and then blocked by incubating in 5% skimmed milk to minimize non-specific binding of antibodies. Blocked blots were submerged with primary antibodies for CD9 and ARF-6 overnight, and subsequently washed three times (10 min each) with 1X Tris buffer saline with 0.1% Tween-20 (TBST) buffer followed by incubation with HRP-conjugated secondary anti-mouse (for CD9) and anti-rabbit (for ARF-6) antibodies. Unbound antibodies were removed by washing with 1X TBST buffer (3 × 10 min), and signal recorded using WestFemto maximum sensitivity substrate kit under Bio-Rad ChemiDoc Imager (Hercules, CA, USA).