The Amido Black stained proteins were excised with a razor blade and N-terminal sequence determination were performed using either ABI model 473A or 477A microsequencers from Applied Biosystems equipped with Problott cartridges.
The spots of interest were excised and soaked two hours in a solution containing acetic acid (100 mM), methanol (10% v/v) and PVP-40 (1% v/v) at 37 C. After three washes in deionized water, the PVDF spots were cut into small pieces (~1 square millimeter) and incubated in 25 microliters of a solution containing sodium phosphate (100 mM) pH 8.0 and lysyl endopeptidase (1 microgram). Following overnight digestion at room temperature, guanidine-HCl (28 mg) and DTT (100 micrograms) were added. After reduction for 2 hours at 37 C, the mixture was incubated for 30 min, at room temperature, with 300 micrograms of iodoacetamide. The digestion solution was removed and guarded. PVDF pieces were then extracted overnight with 25 microliters of a solution containing isopropanol (70% v/v) and trifluoroacetic acid (5% w/v). This elution solution was removed and the PVDF was washed twice with 60 microliters of TFA (0.1% w/v). The digestion and elution solutions were pooled together with two final washes and this mixture was separated by two-dimensional reverse phase HPLC and sequence determination performed.