Mini protean tgx precast 4 20 gradient gels

sEV samples were lysed in non-reducing conditions in Laemmli buffer, heated for 5 min at 95°C, and loaded on mini protean TGX precast 4-20% gradient gels (Biorad) in Tris-glycine buffer for electrophoresis at 110 V for 2 h. Proteins were electro-transferred onto nitrocellulose membranes using the trans-blot turbo transfer system (Biorad) and membranes were blocked with Odyssey blocking buffer (Li-Cor Biosciences) for 1 h. Membranes were then incubated with primary antibodies: mouse anti-CD81 (200 ng/ml, Santa-Cruz), mouse anti-CD63 (500 ng/ml, BD Pharmingen) or mouse anti-CD9 (100 ng/ml, Millipore) overnight at 4°C in Odyssey blocking buffer, followed by incubation with the secondary antibody IRDye 700 goat anti-mouse IgG (Li-Cor Biosciences), for 1 h at room temperature. Membranes were washed three times in TBS 0.1% Tween 20 during 10 min after each incubation step and visualized using the Odyssey Infrared Imaging System (LI-COR Biosciences).

Proteins were separated using a Mini-PROTEAN 3 Electrophoresis System (Bio-Rad). To each sample, an equal volume of Laemmli sample buffer (Bio-Rad) and 2 μL of 2-mercaptoethanol (Sigma-Aldrich) per 50 μL of sample was added. Samples were heated to ~100°C for 5 min and loaded into the wells of Mini-PROTEAN® TGX™ precast 4–20% gradient gels (Bio-Rad) immersed in Tris/glycine buffer (2.5 mM Tris, 19.2 mM glycine, pH 8.3). One well in each gel was loaded with 6 μl of molecular weight marker (Bio-Rad). To allow comparisons between the protein content of samples run on different gels, at least one well per gel contained a protein sample common to all gels (internal standard). Proteins were separated electrophoretically at 100 V for 100 min at room temperature. After separation, the proteins were transferred to nitrocellulose membranes (pore size 0.22 μm; LI-COR, NE, USA) using a Mini Trans-Blot® Electrophoretic Transfer Cell (Bio-Rad). Each tank was filled with Tris/glycine buffer containing 20% (v/v) methanol and transfers were carried out overnight at 30 V and 4°C. After staining with ponceau red (Sigma-Aldrich) to confirm protein transfer was successful, the membranes were washed 3× with distilled water and air dried for 1 h at room temperature.