SDS-PAGE Based Extraction of Extracellular Vesicles Associated Proteins: A Procedure to Extract Proteins from EVs and Prepare Them for In-Gel Digestion
Transcrição
For protein extraction, mix 50 microliters of RIPA buffer with the EV sample for 5 minutes on ice. Sonicate the sample three times at 500 watts and 20 kilohertz for 5 seconds. Then, remove vesicular debris by centrifuging at 20,000 x g for 10 minutes at 4 degrees Celsius.
After isolating the proteins, perform protein migration in the stacking gel of a 12% polyacrylamide gel. Fix the proteins in the gel with Coomassie blue for 20 minutes at room temperature. Then, excise each colored gel piece and cut it into small pieces.
Put the gel pieces through a series of successive washes as described in the manuscript. Then, dry them completely with a vacuum concentrator. After drying, perform protein reduction with 100 microliters of 100-millimolar ammonium bicarbonate containing 10-millimolar dithiothreitol for 1 hour at 56 degrees Celsius.
Next, perform protein alkylation with 100 microliters of 100-millimolar ammonium bicarbonate containing 50-millimolar iodoacetamide for 45 minutes in the dark. Wash the gel pieces according to manuscript directions and completely dry them on the vacuum concentrator.
