Western blot
TBS‐T
WEST
Western blots were used to detect characteristic EV proteins within LEAP‐purified pEVs. Protein extracts from donor platelets and purified EVs were generated with radioimmunoprecipitation assay (RIPA) buffer (Abcam) with a complete protease inhibitor cocktail (Roche). Following sodium dodecyl sulphate‐polyacrylamide gel electrophoresis (SDS‐PAGE), the separated protein was transferred onto polyvinylidene fluoride membranes. The membranes were blocked with 5% non‐fat milk (Biotium) or 3% bovine serum albumin (Sigma) where appropriate, in 20 mM Tris‐HCl (pH 7.5), 500 mM NaCl plus 0.05% Tween (TBS‐T) and immunoblotted at 4°C overnight with primary antibodies: Syntenin (rabbit monoclonal IgG; Cat. no. ab133267, Abcam), CD9 (mouse monoclonal IgG; Cat. no. 312102, Biolegend), CD63 (mouse monoclonal IgG; Cat. no. sc‐5725, Santa Cruz), Calnexin (rabbit polyclonal IgG, Cat. no. ab22595, Abcam). The membranes were extensively washed in TBS‐T and incubated with peroxidase‐conjugated anti‐rabbit IgG antibody (Cat. no. ab6721, Abcam; or Cat. no. 7074S, Cell Signalling Technology) or anti‐mouse IgG antibody (Cat. no. ab6728, Abcam), for 1 h at room temperature. After washing with TBS‐T, the reaction was visualized using ECL detection reagents (SuperSignal™ West Pico PLUS Chemiluminescent Substrate, Thermo Scientific) with a G:Box F3 gel doc system (Syngene).