Srx 101a tabletop processor

Protein from each sucrose gradient fraction (10 in total) obtained after exosome isolation were separated by SDS polyacrylamide gel electrophoresis[7] (link), [30] (link) using a XCell SureLock electrophoresis system (Life Technologies), transferred to Immobilon-FL polyvinylidene difluoride membranes (Millipore, Billerica, MA, USA) and probed with primary mouse monoclonal anti-CD63 (1∶2,000 ab8219, Abcam, Sapphire Bioscience Pty Ltd, NSW, Australia), anti-CD81 (1∶1,500 MAB6435, Abnova, Tapei City, Taiwan) and anti-CD9 (1∶1,500 ab2215, Abcam, Sapphire Bioscience) as previously described exosome enriched markers. [7] (link), [18] (link), [29] , [30] (link) Membranes were washed in Tris buffer saline (pH 7.6) and incubated (1 h) in TBST/0.2% BSA containing horseradish peroxidase–conjugated goat anti-mouse antibody. Proteins were detected by enhanced chemiluminescence with using a SRX-101A Tabletop Processor (Konica Minolta, Ramsey, NJ, USA). The exosomal fractions (from 1.126 to 1.187 g/ml) were pooled (defined as exosomes) and placental alkaline phosphatase (PLAP) protein abundance was determined by Western blot using primary polyclonal antibody anti-PLAP (1∶1,000 ab96588, Abcam, Sapphire Bioscience) using a GS-800 Calibrated Densitometer (Bio-Rad Laboratories).

Dot blotting was performed using the Exo-Check Exosome Antibody Array (Systems Bioscience, CA) as per manufacturer's instructions. Briefly, 500 μg protein equivalent of purified EVs isolated from PC12 culture were lysed and ligated to horseradish peroxidase (HRP) overnight at 4 °C. Ligated protein was then incubated with the antibody membrane for two hours at room temperature. After three washes SuperSignal West Femto Chemiluminescent Substrate kit (Thermo Scientific, UK) was used as per manufacturer's instructions. The blot was visualised via 90 s exposure to X ray film. Film was developed using the Konica SRX-101A Tabletop Processor.