7. DIGE buffer: 65 mM CHAPS, 5 M urea, 2 M thiourea,
0.15 M NDSB-256, 30 mM Tris Base, 1 mM sodium vanadate,
0.1 mM sodium fluoride, and 1 mM benzamidine.
8. LC-MS/MS buffer: 2% SDS, 500 mM Tris Base (pH 7.6),
0.05 M DTT.
2.3 Gel-Based
Proteomics: 2D-DIGE
1. Coomassie plus protein reagent.
2. GE Healthcare CyDye™ DIGE Fluor Minimal Dye Labeling.
3. 10 mM lysine.
4. 2Â sample buffer: 65 mM CHAPS, 2 M thiourea, 5 M urea,
0.15 M NDSB-256, 130 mM DTT, 4 mM tributylphosphine,
1 mM sodium vanadate, 0.1 mM sodium fluoride, and 1 mM
benzamidine.
5. 4–7 Servalyt ampholytes.
6. 2D sample buffer: 5 M urea, 2 M thiourea, 2 mM tributylphosphine, 65 mM DTT, 65 mM CHAPS, 0.15 M NDSB-256,
1 mM sodium vanadate, 0.1 mM sodium fluoride, and 1 mM
benzamidine.
7. pH 4–7 IPG, 24 cm strips.
8. Multiphor II system.
9. Reduction buffer: 6 M urea, 50 mM Tris Base (pH 8.8), 30%
glycerol, 2% w/v SDS, 65 mM DTT, and traces of
bromophenol blue.
10. Alkylation buffer: 6 M urea, 50 mM Tris Base (pH 8.8), 30%
glycerol, 2% w/v SDS, 135 mM iodoacetamide, and traces of
bromophenol blue.
11. 0.5% of melted agarose.
12. 11% polyacrylamide gels.
13. Ettan Dalt 6 system.
14. Typhoon FLA 7000 scanner.
15. Fixing buffer: 10% methanol/7% acetic acid.
16. SYPRO Ruby Protein Gel Stain.
17. Progenesis SameSpots software (v 4.5).
18. EASY-nLC Proxeon.
19. Bruker Amazon ETD ion trap.
20. Easy column SC200 Proxeon.
21. Data Analysis 4.0 and BioTools 3.2.
22. Mascot v2.3.0 database.
23. SwissProt human database.
Circulating EVs Clinical Proteomics
15