Overview
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% Nonidet P-40, 1 mM Phenylmethanesulfonyl fluoride [PMSF], 1x protease inhibitor) with the EV sample (25 μL resulting from the EV concentration on filter) for 5 min on ice to extract proteins.
- Sonicate for 5 s (amplitude: 500 W; frequency: 20 kHz), 3 times on ice.
- Remove vesicular debris by centrifugation at 20,000 x g for 10 min, at 4 °C.
- Collect the supernatant and measure the protein concentration with the Bradford protein assay method.
- Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and western blotting
- Mix protein extracts (30 μg) with 5c Laemmli sample buffer (v/v).
- Load the protein mix on a 12% polyacrylamide gel.
- Migrate the proteins in the gel with TGS buffer (25 mM Tris pH 8.5, 192 mM Glycine, and 0.1% SDS), at 70 V for 15 min and 120 V for 45 min.
- Transfer the proteins onto a nitrocellulose membrane with semi-dry system at 230 V for 30 min.
- Saturate the membrane for 1 h at RT with blocking buffer (0.05% Tween 20 w/v, 5% milk powder w/v in 0.1 M PBS, pH 7.4).
- Incubate the membrane overnight at 4 °C with mouse monoclonal anti-human heat-shock protein 90 (HSP90) antibody diluted in blocking buffer (1:100).
- Wash the membrane three times with PBS-Tween (PBS, 0.05% Tween 20 w/v) for 15 min.
- Incubate the membrane for 1 h at RT with goat horseradish peroxidase-conjugated anti-mouse IgG secondary antibody diluted in blocking buffer (1:10,000).
NOTE: A negative control is performed using secondary antibody alone. - Repeat the washing step (step 1.1.2.7).
- Reveal the membrane with enhanced chemiluminescence (ECL) western blotting substrate kit (see Table of Materials).
- Protein extraction
2. Proteomic Analysis
- Protein extraction and in-gel digestion
- Isolate the EVs by size exclusion chromatography column (SEC) and quantify using nanoparticle tracking analysis (NTA). Repeat step 1.1.1 for EV protein extraction.
- Perform protein migration in the stacking gel of a 12% polyacrylamide gel.
- Fix the proteins in the gel with Coomassie blue for 20 min at RT.
- Excise each colored gel piece and cut it into small pieces of 1 mm3.
- Wash the gel pieces successively with 300 μL of each solution: ultrapure water for 15 min, 100% acetonitrile (ACN) for 15 min, 100 mM ammonium bicarbonate (NH4HCO3) for 15 min, ACN:100 mM NH4HCO3 (1:1, v/v) for 15 min, and 100% ACN for 5 min with continuous stirring.
- Dry completely gel pieces with a vacuum concentrator.
- Perform protein reduction with 100 μL of 100 mM NH4HCO3 containing 10 mM dithiothreitol for 1 h at 56°C.
- Perform protein alkylation with 100 μL of 100 mM NH4HCO3 containing 50 mM iodoacetamide for 45 min in the dark at RT.
- Wash the gel pieces successively with 300 μL of each solution: 100 mM of NH4HCO3 for 15 min, ACN:20 mM NH4HCO3 (1:1, v/v) for 15 min, and 100% ACN for 5 min with a continuous stirring.
- Completely dry the gel pieces with a vacuum concentrator.
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Materials
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 |