Page ruler prestained protein size markers

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).

A total of 15 ng of each CleavEx protein was incubated with 50, 250, and 500 nM KLK13 in 50 mM tris (pH 7.5). For the full-length S protein, fractions containing purified HKU1-S or mock samples were incubated with 0.5, 1.0, or 5.0 μM KLK13 in 50 mM tris (pH 7.5). Samples were incubated at 37°C for 3 hours, and the reactions were stopped with the addition of 50 mM dithiothreitol (DTT)–supplemented SDS sample buffer (1:1), boiled for 5 min, cooled on ice, and resolved on 10% SDS-PAGE gels together with dual-color Page Ruler Prestained Protein size markers (Thermo Fisher Scientific, Poland). The separated proteins were then transferred onto a Westran S PVDF membrane (GE Healthcare, Poland) by wet blotting (Bio-Rad, Poland) for 1 hour at 100 V in transfer buffer (25 mM tris, 192 mM glycine, and 20% methanol) at 4°C. The membranes were then blocked by overnight incubation at 4°C in TBS-Tween (0.1%) buffer supplemented with 5% skimmed milk (BioShop, Canada). An HRP-labeled anti–His tag antibody (1:25,000 dilution; Sigma-Aldrich, Poland) diluted in 5% skimmed milk/TBS-Tween (0.1%) was used to detect the His-tagged HmuY proteins. The signal was developed with the Pierce ECL Western blotting substrate (Thermo Fisher Scientific, Poland), and bands were visualized with the ChemiDoc Imaging System (Bio-Rad, Poland).