**2.8 Isolation of protein spots**

Protein spots were excised from the gels using a syringe and transferred to eppendorf tubes. For silver destaining, 25 µL of 100 mM sodium thiosulphate and 25 μL of potassium ferricyanide were added to the gel pieces (Gharahdaghi et al., 1999). When the pieces were completely destained, the chemicals were removed by washing (6×5 min with Milli-Q water) before addition of 50 μL of 200 mM ammonium bicarbonate and incubation for 20 min at room temperature. The gel pieces were washed (3x5 min with Milli-Q water) and dehydrated with 100% acetonitrile (ACN) for 5 min or until the gel pieces were opaque white. After removal of the ACN, the gel pieces were dried in a SpeedVac vacuum concentration system (Savant, Farmingdale, NY). Protein spots excised from Sypro Ruby stained gels were washed in 50% ACN/25 mM ammonium bicarbonate 2x30 min prior to dehydration with 100% ACN.
