Gas phase sequencer model 492

Recombinant p.W435R variant of C1r was produced in the FreeStyle 293 Expression System (Thermo Fisher), using a pcDNA3.1/Neo(+) plasmid encoding human C1r with a C-terminal Strep-tag. 293-F cells grown in FreeStyle 293 medium were transfected with this plasmid using 293 fectin and stable transfectants were selected with 400 μg/ml neomycin (Thermo Fisher). Recombinant C1r was purified from the culture supernatant by chromatography on StrepTactin Sepharose High performance (Sigma-GE28-9355-99), as recommended by the manufacturer. Briefly, the cell culture supernatant was concentrated 6-fold, dialyzed against 100 mM Tris, 150 mM NaCl, 1 mM EDTA, pH 8.0, and applied to a 1-ml column equilibrated in the same buffer. Elution was carried out using 2.5 mM desthiobiotin (Sigma-Aldrich) in the same buffer. The eluted protein was dialyzed in the same buffer and concentrated to 0.3–0.45 mg/ml. N-terminal sequence determination of the major band observed after SDS-PAGE analysis under non-reducing conditions was performed using an Applied Biosystems gas-phase sequencer model 492 coupled online with an Applied Biosystems Model 140C HPLC system.

Recombinant VE-cadherin was exposed to purified LasB and reaction products were electrophoresed, transferred on PVDF membrane and stained with Coomassie. The 50 kDa band was excised 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. Phenylthiohydantoinamino 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 Model 610A. 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

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).