1. CUTING THE SPOTS
Cut the protein bands with a scalpel and use the plastic tip of a pipette (previously cut) to remove the spots. Deposit each one in a 96-multiwell plate or in eppendorf tubes .
2. WASHING
Make 2 washing cycles 5 min/each with Milli-Q water.
3. DESTAINING
- Prepare the destaining solution: 30 mM potassium ferricyanide (K 3 Fe(CN) 6 ) (9,9 mg/ml) and 100 mM sodium thiosulfate (Na 2 S 2 O 3 ) (24,8 mg/ml).
- Cover the bands with the destaining solution for 10 min. Carefully remove the destaining solution and discard.
4. WASHING
Make 2 washing cycles 5 min/each with Milli-Q water.
5. DEHYDRATION
- Add acetonitrile (AcN) to cover the band or spot (~ 20 microliters) and dehydrate the gel for 5 min.
- Repeat this operation twice or until the gel has an opaque white colour and a significantly smaller size.
6. DRY in a vacuum centrifuge.
7. REDUCTION And ALKYLATION
- Cover the bands with a 10 mM Dithiothreitol (DTT) solution in 25 mM ammonium bicarbonate (NH 4 HCO 3 ) for 30 min at 56°C. Leave at room temperature. Carefully remove the DTT and discard.
- Add AcN. Carefully remove the AcN and discard.
- Quickly add 55mM Iodoacetamide (10 mg/mL) in 25 mM NH 4 HCO 3 and incubate for 15 min in the dark.
8. WASHING
- Add AcN for 5 min. Carefully remove the AcN and discard.
- Add 25 mM NH 4 HCO 3 for 5 min. Without removing the buffer, add an equal volume of AcN. Incubate for 15 min. Carefully remove the supernatant and discard.
9. DRY in a vacuum centrifuge.
10. DIGESTION
- Prepare a 12.5 ng/ml trypsin solution in 25 mM NH 4 HCO 3 . Keep on ice until use.
- Add the trypsin. Incubate on ice for 45 min to rehydrate the gel pieces (trypsin solution will be absorbed by the gels but no activity will occur).
- Carefully remove the excess of trypsin and discard.
- Add 25 mM NH 4 HCO 3 until covering the bands.
- Incubate overnight (or at least 6 hours) at 37°C.
-Gently vortex the samples and centrifuge them. Collect the supernatant and transfer the sample to a new multiwell plate or tube.
11. PEPTIDE EXTRACTION
- Cover the gel pieces with a 50% AcN/0.5% TFA solution and sonicate for 10 min. Collect the supernatant and transfer the sample to a new multiwell plate or tube.
Repeat this operation 3 times and combine the supernatants of each extraction.
- Cover the gel pieces with AcN for 10. Collect the supernatant. Repeat this operation 3 times and combine the supernatants of each extraction.
12. COMBINE ALL SUPERNATANTS and dry the sample in a vacuum centrifuge. Dissolve in 5 microliter of a 50% AcN/0.1% TFA solution.
