Table 3 Optimized protocol for protein extraction
From: Systematic evaluation and optimization of protein extraction parameters in diagnostic FFPE specimens
1. Start with 2 tissue Sects. (10 µm) on glass slides or directly in tube |
2. Incubate in xylol (100%) for 15Â min |
3. Discard xylol |
4. Repeat once from step 2 |
5: Immerse briefly in ethanol (70 % v/v) to remove residual xylol |
6. Immerse slides in double-destilled water for 30Â s |
7. Tap slides on paper towel to remove water, do not let dry |
8. Scratch tissue into 0.2Â ml PCR tube |
9. Add 100 µl buffer S (SDS 2% (w/v), Tris-base 200 mM, EDTA 1 mM, pH 7.2) [µl] |
10. Add 5 µl beta-mercaptoethanol [µl] |
11. Add 1 µl proteinase & phosphatase inhibitor 1005 (ThermoFisher 78840) [µl] |
12. Incubate on ice for 5Â min |
13. Vortex |
14. Place tubes in a thermocycler: |
15. 4 °C for 5 min |
16. 90 °C for 90 min |
17. 99 °C for 5 min |
18. 60 °C for 10 min |
19. Repeat 4 times from step 17Â Step 16 |
20. Keep at 4° C if not processed directly |
21. Vortex |
22. Centrifuge at 4 °C, 10,000×g for 15 min |
23. Save supernatant (extract) |