SDS-PAGE Based Extraction of Extracellular Vesicles Associated Proteins: A Procedure to Extract Proteins from EVs and Prepare Them for In-Gel Digestion
In this video, we demonstrate the SDS-PAGE in-gel digestion method for protein extraction from extracellular vesicles or EVs. Once isolated, the obtained protein fragments can be used for proteomic analysis.
Protocol
1. Characterization of EVs Western Blot Analysis Protein extraction Isolate the EVs by size exclusion chromatography column (SEC) and quantify using nanoparticle tracking analysis (NTA). Mix 50 μL of RIPA buffer (150 mM sodium chloride [NaCl], 50 mM Tris, 5 mM Ethylene glycol-bis(2-aminoethylether)-N,N,N′,N′-tetraacetic acid [EGTA], 2 mM Ethylenediamine-tetraacetic acid [EDTA], 100 mM sodium fluoride [NaF], 10 mM sodium pyrophosphate, 1% …
Divulgations
The authors have nothing to disclose.
Materials
Acetonitrile
Fisher Chemicals
A955-1
Ammonium bicarbonate
Sigma-Aldrich
9830
HSP90 α/β antibody (RRID: AB_675659)
Santa-cruz
sc-13119
Bio-Rad Protein Assay Dye Reagent Concentrate (Bradford)
SDS-PAGE Based Extraction of Extracellular Vesicles Associated Proteins: A Procedure to Extract Proteins from EVs and Prepare Them for In-Gel Digestion. J. Vis. Exp. (Pending Publication), e20624, doi: (2023).