1. Run samples at 150 V until the samples run 1 cm into the separating gel
2. 10 min Destainer (40% ethanol, 7% acetic acid); 5 min Destainer; 2x 5 min H2O
3. Colloidal Coomassie 16 h
4. 2x 5 min in 1% acetic acid
5. Cut the bands and cut in 1 mm x1 mm pieces, put in 1.5 mL tube
6. Destain
7. Wash the gel pieces with ~200 µL 40 mM ammonium bicarbonate (ABC) for 5 min (with shaking, 900rpm (should be turbulent shaking!) at ambient temperature. Discard liquid into waste.
8. Incubate gel pieces in 70% acetonitrile (in water) for 15 min (with shaking as before) at ambient temperature. Gel may go cloudy or opaque as it becomes dehydrated. Discard liquid into waste.
9. Repeat steps 2 and 3 two more times (until the gel slice is completely (nearly) colorless).
10. Completely dehydrate gel by adding 100% acetonitrile and let it stand for at least 5 minutes. Discard liquid into waste.
11. Dry gel pieces under vacuum (~5 min). Now you can store them at -20°C
12. Prepare enough Trypsin to digest all samples. Prepare 10 ng/uL proteomic grade trypsin in 40 mM ABC.
13. Add 30 - 50 μL (depending on the amount of protein) of the enzyme solution from step 12. to each tube. Let tubes stand in ice for 20-30 minutes. Add more 40mM ABC Let sit for another 5 min, then remove excess enzyme solution (if it is really too much).
14. Add 50-100 μL (enough to cover all) 40 mM ABC to each tube and incubate at 37° C for at least 3 h (tubes can be left o/n at this stage).
15. Spin the liquid briefly down and add 2% Formic acid
16. 20 min shaking as before, transfer liquid to a high quality 1.5 mL tube
17. Add 100µl 2%FoAc, 10% ACN to gel pieces, shake 20 min. This extraction will contain the more hydrophilic peptides.
18. Remove the solution and add to the eluate tube.
19. To the gel piece add 200 μL of 60% acetonitrile containing 1% formic acid, shake for >20 min. This wash will contain most of the tryptic peptides. Add this supernatant to the third extraction.
20. Speed vac the samples, first only 25 mbar 20 min, than go down to 2.5 mbar. As soon as the first tubes are “empty” remove them, keep them at 4°C for a short time or -20°C for a longer time.
