Supporting information:
IEF Protocol:
Step1: 0-200 V, 1 min
Step 2: 200 V, 2 h
Step 3: 200-500 V, 1 min
Step 4: 500 V, 2 h
Step 5: 500-1000 V, 5 min
Step 6: 1000 V, 1,5 h
Step 7: 1000-4000 V, 30 min
Step 8: 4000 V, 12 h
Step 9: 1000 V, 20 h
2D-Gel picture from E. coli lysate with the 20 spots cut, both identical for manual and Oasis.
Detailed protocol as used here for manual digestion of Coomassie Blue stained gels
needed solutions:
 100 mM ammonium bicarbonate in water (HPLC grade), pH 8.0
o (316 mg NH4HCO3 + 40 mL H2O)
 acetonitrile (HPLC grade)
 10 mM dithiothreitol in 100 mM ammonium bicarbonate
o (1.5 mg DTT/1 mL 100 mM NH4HCO3)
 55 mM iodoacetamide (IAA) in 100 mM ammonium bicarbonate
o (10 mg iodoacetamide/1 mL 100 mM NH4HCO3)
 8 µL aliquots of trypsin (modified, sequencing grade) in 1 mM HCl (0.1 µg/µL)
 digestion solution (5 samples):
extraction solution:
(v/v) :
50 µL (100 mM NH4HCO3)
50 µL H2O
8 µL trypsin stock solution (0.1 µg/µL)
acetonitrile
Water HPLC grade
formic acid
47.5 %
47.5 %
5%
excision of protein bands (spots) from gels
1. rinse the entire gel with water and excise bands of interest with a clean scalpel,
cutting as close to the edge of the band as possible
2. chop the excised bands into cubes (1x1 mm)
3. transfer gel pieces into a micro-centrifuge tube (preferably 0.5 mL tubes) and label
them in blue color
4. spin down and remove all liquid
5. add acetonitrile (the volume should be at least twice the volume of the gel pieces)
6. shake the gel pieces for 10 to 15 min until they have shrunk (they become white and
small and start sticking together)
7. remove all liquid
destaining and reduction
8. rehydrate gel pieces in 10-50 µL of 100 mM NH4HCO3
9. add an equal volume acetonitrile and shake for 10-15 min until the gel pieces have
destained
10. remove all liquid and add 10-50 µL acetonitrile to shrink the gel pieces
11. remove all liquid and swell gel pieces in 10 mM DTT in 100 mM NH4HCO3 (the
reducing buffer should cover the gel pieces completely) and incubate for 30 min at
56°C to reduce cysteine bridges of the protein
12. spin gel pieces down and remove excess liquid
13. shrink gel pieces with acetonitrile and wait for 10-15 min until the gel pieces have
shrunk
14. spin gel pieces down and remove all liquid
Alkylation
15. swell gel pieces with 55 mM IAA in 100 mM NH4HCO3 and incubate for 20 min at
room temperature in the dark
16. remove IAA solution and wash gel pieces with 20-50 µL of 100 mM NH4HCO3 for 15
min
17. remove NH4HCO3 , add 10-50 µL acteonitrile and wait 10 min for shrinking
application of trypsin
18. remove all liquid, rehydrate gel pieces in the digestion buffer (15-20 µL for small gel
pieces like from a spot cutter) at 4°C (use ice bucket)
19. after 30 min remove remaining buffer
20. add 10-20 µL of the same buffer but prepared without trypsin to cover gel pieces and
keep them wet during enzymatic digestion
21. leave samples in a heating block at 37°C overnight
extraction of peptides (from now on never discard a solution)
22. copy the blue label of each tube in RED COLOR onto a NEW 0.5 mL Eppendorf tube
23. take samples from the heating block, centrifuge gel pieces down and collect the
digestion solution into the corresponding red labeled tube
24. add to the gel pieces 10-20 µL extraction solution (3 times the volume of the gel
pieces)
25. place blue labeled tubes with gels (optional at 37oC) for 15 min in a sonication bath
26. centrifuge gel pieces down and collect the extraction solution again into the
corresponding red labeled tube
27. add to blue labeled tubes with gels 10-20 µL acetonitrile (2 times the volume of the
gel pieces) and place the tubes for 15 min in a sonication bath
28. centrifuge gel pieces down and collect the acteonitril again into the corresponding red
labeled tube
29. dry the pooled extracts (red labeled tube) using a vacuum concentrator ("speed-vac")
at 30°C (do not let the peptides run dry for a longer time)
30. freeze the dried extracts (peptides) at -20 oC or -80 oC (for > 1 month)