Procise pc v2

Limited proteolysis of HTNV-NC was performed at 20°C in lysis buffer with a 3:2 w/w ratio of HTNV-NP/trypsin. Proteolysis was stopped by the addition of denaturing SDS-PAGE loading dye and incubation at 95°C for 5 min. Digestion was visualised in 10% acrylamide SDS-Page gels. For N-terminal sequencing, proteins were transferred on PVDF membrane previously incubated in 10 mM CAPS pH 11, 10% methanol buffer. PVDF membrane was stained using 0.1% Coomassie Brilliant Blue R-250, 40% methanol, 1% acetic acid buffer until bands were visible, washed using 50% methanol and dried. Visible bands were cut and subjected to N-terminal sequencing. Amino acid sequence determination based on Edman degradation was performed using an Applied Biosystems gas-phase sequencer model 492 (s/n: 9510287J). Phenylthiohydantoin amino acid derivatives generated at each sequence cycle were identified and quantitated on-line with an Applied Biosystems Model 140C HPLC system using the data analysis system for protein sequencing from Applied Biosystems (software Procise PC v2.1).

Amino acid sequence determination based on Edman degradation was performed using an Applied Biosystems gas-phase sequencer model 492 (s/n: 9510287J). Phenylthiohydantoin amino acid derivatives generated at each sequence cycle were identified and quantitated on-line with an Applied Biosystems Model 140C HPLC system using the data analysis system for protein sequencing from Applied Biosystems (software Procise PC v2.1). The PTH-amino acid standard kit (Perkin-Elmer P/N 4340968) was used and reconstituted according to the manufacturer’s instructions. The procedures and reagents used were as recommended by the manufacturer. Chromatography was used to identify and quantify the derivatized amino acid removed at each sequence cycle. Retention times and integration values of peaks were compared to the chromatographic profile obtained for a standard mixture of derivatized amino acids.