This article outlines a method for protein extraction from extracellular vesicles (EVs) using lysis buffers and SDS-PAGE. The process includes sonication, centrifugation, and various chemical treatments to prepare proteins for proteomic analysis.
To begin, take a tube containing intact extracellular vesicles or EVs.
Suspend the EVs in the desired volume of lysis buffer and sonicate the sample. This treatment lyses the EV membrane and shears the genetic material, releasing the proteins into the buffer.
Centrifuge to pelletize the cell debris and sheared genetic material. Collect the supernatant containing various proteins.
Add a loading dye to the protein extract and run it on an SDS-PAGE gel.
Soak the gel in a staining solution to allow the dye molecules to bind proteins. Locate the stained band containing the desired protein and excise it.
Cut the colored band into small pieces to improve the accessibility of proteins to enzymes in the subsequent steps. Transfer these pieces into a microcentrifuge tube.
Add a destaining solution that removes the staining dye. Remove the spent destaining solution.
Add a reduction solution. Incubate to allow the reducing agent to cleave the disulfide bonds in protein, exposing the free sulfhydryl groups.
Treat the reduced proteins with an alkylating solution. The alkyl groups bind to the free sulfhydryl groups, keeping the protein unfolded.
Discard excess solution and wash the pieces with a dehydrating solution to remove the alkylating reagent. Dry the gel completely.
Gel pieces are now ready for further proteomic analysis.
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.
繁體中文
