SDS-PAGE Based Extraction of Extracellular Vesicles Associated Proteins: A Procedure to Extract Proteins from EVs and Prepare Them for In-Gel Digestion

Abstract

Source: Lemaire, Q. et al. Characterization of Immune Cell-derived Extracellular Vesicles and Studying Functional Impact on Cell Environment. J. Vis. Exp. (2020)

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

1. Western Blot Analysis
   1. Protein extraction
      1. Isolate the EVs by size exclusion chromatography column (SEC) and quantify using nanoparticle tracking analysis (NTA).
      2. 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% Nonidet P-40, 1 mM Phenylmethanesulfonyl fluoride [PMSF], 1x protease inhibitor) with the EV sample (25 μL resulting from the EV concentration on....

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
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)	Bio-Rad	5000006
Dithiothreitol	Sigma-Aldrich	43819
Ethylene glycol-bis(2- aminoethylether)-N,N,N′,N′- tetraacetic acid	Sigma-Aldrich	03777-10G
Ethylenediaminetetraacetic acid	Sigma-Aldrich	ED-100G
Fisherbrand Q500 Sonicator with Probe	Fisherbrand	12893543
Idoacetamide	Sigma-Aldrich	I1149
InstantBlue Coomassie Protein Stain	Expedeon	ISB1L
Laemmli 2x	Bio-Rad	1610737
Nonidet P-40	Fluka	56741
Peroxidase AffiniPure Goat AntiMouse IgG (H+L)	Jackson ImmunoResearch	115-035-003
Phalloidin-tetramethylrhodamine conjugate	Santa-cruz	sc-362065
Protease Inhibitor	Sigma-Aldrich	S8830-20TAB
Sodium Chloride	Scharlau	SO0227
Sodium Dodecyl Sulfate	Sigma-Aldrich	L3771
Sodium Fluoride	Sigma-Aldrich	S7920-100G
Sodium pyrophosphate	Sigma-Aldrich	S6422-100G
SpeedVac Vacuum Concentrator	ThermoFisher
Trans-Blot SD Semi-Dry Transfer cell	Bio-Rad	1703940