Pierc bca protein assay kit

Concentration of proteins in samples was determined using the Pierc™ BCA Protein Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s instructions, with BSA as a standard. Each measurement was performed in duplicate at 562 nm absorbance.

Cells and exosomes were lysed in radioimmunoprecipitation assay (RIPA) buffer (Pierce® 899000; Thermo Fisher Scientific) and protease cocktail inhibitor‐EDAT free (cOmplete Mini, #11836170001; Sigma–Aldrich). Protein concentration was determined by Pierc ™ BCA protein assay kit (#23225; Thermo Fisher Scientific). Approximately 20 µg protein was loaded on 10% SDS‐PAGE and then the separated proteins were blotted onto nitrocellulose membrane (Thermo Fisher Scientific). The membranes were washed three times with Tris buffered saline containing 0.1% of tween 20 (TBST) pH 6.5 for 10 min/washing step followed by masking with 5% BSA for 1 h. Membrane development was performed by incubating the primary antibodies (ALIX, CD9, CD81, CD63, flotillin‐1, TSG101, Grp94, promini‐2 and β‐actin) with the recommended dilution from suppliers overnight at 4°C. The membranes were washed three times (10 min each) with TBST and then the membranes were incubated with the corresponding secondary antibodies conjugated to HRP for 1 h at ambient temperature. Following washing steps with TBST, target proteins were detected using an enhanced chemiluminescence kit SuperSignal^Tm West Pico Plus (Thermoscientific, Rockford, USA).