Ppsq 31a automatic protein sequencer

A total of 10 μg of S1/S2 CleavEx protein was incubated with 500 nM KLK13 at 37°C for 5 hours. The reaction was stopped by the addition of 50 mM DTT-supplemented SDS sample buffer (1:1), and samples were immediately boiled for 5 min. The samples were then resolved on 10% SDS-PAGE gel together dual-color Page Ruler Prestained Protein size markers (Thermo Fisher Scientific, Poland) in the Tris-Tricine SDS-PAGE system. The separated proteins were then electrotransferred onto a Western S PVDF membrane with the Trans-Blot SD Semi-Dry Transfer Cell (Bio-Rad, Poland). The transfer was performed for 30 min at 15 V in transfer buffer [10 mM N-cyclohexyl-3-aminopropanesulfonic acid and 10% methanol (pH 11)]. The membranes were then stained with 0.025% (w/v) Coomassie Brilliant Blue R-250 (BioShop, Poland), and proteins in the bands of interest were sequenced by Edman degradation with a PPSQ-31A automatic protein sequencer (Shimadzu, Japan).