Coomassie brilliant blue r 250

Proteins were extracted from the same samples harvested for viral RNA quantification, above. Extractions from buffer RLT were performed using the iced acetone method described by the manufacturer (Qiagen). Proteins were separated by denaturing SDS polyacrylamide gel electrophoresis, and transferred to PVDF (Pall Corp., Pensacola, FL). Immunoblotting was performed with monoclonal antbody to influenza NP (AA5H; AbCam, Cambridge, MA) or anti-M1 polyclonal (a kind gift of Dr. Adolfo García-Sastre, Icahn School of Medicine at Mount Sinai), and peroxidase conjugated secondary antiserum. Blots were imaged with Supersignal substrate (ThermoFisher Scientific, Carlsbad, CA), on a Cell Biosciences FluorChem HD2. Consistent loading was monitored by Coomassie Brilliant Blue R-250 (Amresco, Solon, OH) staining of the post-transfer gel.

The transgenic milk samples were diluted with a 2-fold volume of EDTA (Sigma, St. Louis, MO, USA) at a final concentration of 20 mM to extract rhGH within casein micelles. The treated milk was centrifuged at 11,000 × g for 1 h at 4°C. Defatted milk samples were acidified by slowly adding 50% acetic acid (Merck Millipore, Billerica, MA, USA) with constant stirring until the milk reached pH 4.25, which precipitated casein. The samples were then centrifuged at 11,000 × g for 1 h at 4°C. The whey samples were neutralized using 2 M Tris (AMRESCO, Solon, OH, USA) and filtered through a 0.2-μm hollow fiber membrane microfilter (GE Healthcare Life Sciences, Little Chalfont, UK). Samples containing rhGH were clarified by tangential flow filtration with a nominal 300-kDa pore size hollow fiber membrane ultrafilter (GE Healthcare Life Sciences, Little Chalfont, UK). The 300-kDa filter permeate was concentrated and diafiltered through a nominal 5-kDa cut-off size hollow fiber membrane ultrafilter (GE Healthcare Life Sciences, Little Chalfont, UK). The supernatant from each step was analyzed by SDS-PAGE using 13.5% (w/v) gel under reducing conditions. Proteins were stained with Coomassie brilliant blue R-250 (AMRESCO, Solon, OH, USA).

The activities of the expressed wild type and mutant KLK4 proteins were assayed using gelatin zymography. Thermolysin (Sigma-Aldrich, St. Louis, MO, USA) was dissolved in a buffer containing 50 mM Tris and 5 mM CaCl₂ at a concentration of 20 µg/mL. Then, 10 µL of concentrated media was mixed with 1 µL of Thermolysin solution, and the mixture was incubated at 37 °C for 16 h. Samples were mixed with 5× non-reducing loading buffer and electrophoresed on 12% SDS-PAGE gel containing 0.1% gelatin (Sigma-Aldrich) at 80 V for 5 h in an ice box. After electrophoresis, the gel was incubated in 1× renaturing buffer (Novex, Waltham, MA, USA) at room temperature for 15 min and this step was repeated 3 times. After the renaturing step, the gel was incubated at 37 °C for 24 h in 1× developing buffer (Novex). The zymogram was stained with 0.5% Coomassie brilliant blue R-250 (Amresco, Radnor, PA, USA), dissolved in 45% MeOH, 10% acetic acid staining solution for 1 h and finally visualized after washing with destaining solution (25% EtOH, 10% acetic acid).