Ponceau S and western blot

Protein concentrations of individual eluate fractions obtained from 3 different plasma samples were measured by bicinchoninic acid assay (BCA) according to manufacturer’s instructions. Samples were then either concentrated or diluted to have a concentration value within the range of 300 µg/ml and 2000 µg/ml. Equal protein amount of each sample was mixed with reducing 5 × Laemmli sample buffer (Elpis-Biotech) and heated for 3 min at 95 °C. Proteins were then separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) by using 4–20% gradient polyacrylamide gels (Bio-Rad, Cat #: 456-1093). Membranes were transferred to a nitrocellulose membrane by using Trans-Blot Turbo system (Bio-Rad). Afterwards, the nitrocellulose membranes were stained with Ponceau S solution for visualizing protein bands by using ChemiDoc imaging system (Bio-Rad). For subsequent immunostaining, the membranes were first blocked with 5 w/v% skim milk in Tris-buffered saline supplemented with 0.1% Tween-20 (TBST) for an hour and incubated with the following antibodies overnight at 4 °C: anti-ApoB (1:2000, Santa Cruz Biotechnology, sc-25542), anti-CD9 (1:1000, Santa Cruz Biotechnology, sc-9148), anti-CD63 (1:1000, Bioss, bs-1523R), anti-CD81 (1:1000, Bioss, bs-6934R), and anti-albumin (1:8000, Bioss, bs-0945R). After 5 washes with TBST, the membranes were incubated with horseradish peroxidase conjugated anti-rabbit secondary antibodies for 2 h at room temperature (1:2000, Cell Signaling Technology, 7074S). The membranes were then washed 5 times with TBST and incubated with Clarity western ECL buffer (Bio-Rad, Cat #: 170-5060) for 15 min. Finally, the immunoreactive bands were visualized by ChemiDoc imaging system (Bio-Rad). Intensities of bands were analyzed by using Image Lab Software 5.2 (Bio-Rad).

For data analysis, the intensities of protein bands were compared with the band intensity with the highest value. Specifically, the band with the highest intensity was set to 1. Western blot intensities were not normalized by the Ponceau S staining to avoid staining bias, which might occur upon loading insufficient amount of proteins (Supplementary Fig. ^S19a). To ensure the controlled loading, Western blot results were normalized against Ponceau S staining only in case that the sufficient amount of proteins were present. No significant difference in elution trends was noted when the normalized Western blot results were compared with its unnormalized counterpart (Supplementary Fig. ^S19b). Although the performed analysis method was confirmed that there is no effect in elution trends of Western blot, there remains a limitation in that only approximate overall purity can be confirmed.