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.
1. Characterization of EVs
2. Proteomic Analysis
The authors have nothing to disclose.
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 |