4.7. Characterization of Small Extracellular Vesicles with Western Blot
EV-associated antigens
Western blot was performed to characterize the expression of small EV-associated antigens in small EVs isolated with ultracentrifugation and Exoquick, in comparison to MVs and plasma. Samples (1 μg) were mixed with Laemmli buffer (with β-mercaptoethanol for albumin and without β-mercaptoethanol for CD9, CD63 and CD81) and boiled at 90 °C for 10 min to denature and reduce the samples. Afterwards samples and a prestained protein ladder as reference (26616, Thermo Fisher Scientific, Waltham, MA, USA) were applied to the wells of a 10% BOLT bis-tris plus gel (NW00102BOX, Thermo Fisher Scientific, Waltham, MA, USA) and electrophoresis was performed in an XCell SureLock Mini-Cell Electrophoresis System (Thermo Fisher Scientific, Waltham, MA, USA) with 1× MOPS SDS running buffer (13226499, Thermo Fisher Scientific, Waltham, MA, USA) at 200 V, 90 W, 530 mA for 45 min. After electrophoresis, the gel was allowed to equilibrate in 1× BOLT transfer buffer (BT0006, Thermo Fisher Scientific, Waltham, MA, USA) with 20% methanol (10284580, Fisher Scientific, Schwerte, GER) for 10 min. Protein transfer onto 0.2 μm PVDF membranes (1620177, Bio-Rad Laboratories, Hercules, CA, USA) was performed in BOLT transfer buffer with 20% methanol at 30 V, 100 mA, 90 W for 16 h. To assess successful transfer, the membrane was stained with the Revert 700 total protein stain kit (926-11010, LI-COR Biosciences, Lincoln, NE, USA) and the gel was stained with SimplyBlue SafeStain (LC6060, Thermo Fisher Scientific, Waltham, MA, USA). As only 1 μg of protein was available for Western blot analysis, SuperSignal™ Western Blot Enhancer (46640, Thermo Fisher Scientific, Waltham, MA, USA) was used according to the manufacturer’s protocol. Blocking of the membrane was performed in 5% milk in 1× TBST for 1 h at room temperature. Incubation with primary antibodies was performed overnight. A list of used primary antibodies, as well as dilutions, can be found in 