In-gel digest for silver-stained gels
- Protein spots are excised, placed in Eppendorf tubes, washed with MilliQ and shrunk by dehydration in acetonitrile
- Gel pieces are dried in vacuum centrifuge
- Gel pieces are completely destained in 30 mM K3[Fe(CN)6] / 100 mM Na2S2O3 : 1/1 for 10’ and washed afterwards with MilliQ
- Gel pieces are shrunk again by addition of acetonitrile and dried in vacuum centrifuge
- Gel pieces are swollen in a digestion buffer (12.5ng trypsin/µl 50 mM NH4HCO3) at 4°C (on ice)
- After 45’ the supernatant is replaced with 50 mM NH4HCO3
- Enzymatic cleavage : 37°C, overnight
- Peptide extraction : 1/3 20mM NH4HCO3, 2/3 acetonitrile (5 h)