SDS-PAGE Based Extraction of Extracellular Vesicles Associated Proteins: A Procedure to Extract Proteins from EVs and Prepare Them for In-Gel Digestion
Published: April 30, 2023
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 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% …
Disclosures
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) | 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 |
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Cite This Article
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).