Bio safe cbb

Purified virus-like particles were subjected to viral dsRNA extraction and analysis as previously described^{23 (link)}. Briefly, 200 μl sucrose suspension was collected from each fraction after sucrose gradient centrifugation and treated with phenol/chloroform/isoamyl alcohol (25:24:1) (pH 5.2) to remove viral proteins. The nucleic acids were precipitated with ethanol, dissolved in DEPC-treated water and analyzed by agarose gel electrophoresis.
Proteins extracted from each sucrose fraction were analyzed by 12% SDS-PAGE with 25 mM Tris-glycine and 0.1% SDS. Following electrophoresis, the gels were stained with Coomassie brilliant blue R-250 (Bio-Safe CBB; Bio-Rad, USA). The protein bands on the gel were then individually excised and subjected to PMF analysis by Sangon Biotech (Shanghai) Co., Ltd, China, as previously described^{27 (link)}.

Synthesized and purified QconCAT proteins were separated on a NuPAGE 4–12% Bis-Tris gel (Life Technologies), and stained using Bio-Safe CBB (Bio-Rad, Hercules, CA). Protein bands were excised from the gel, and each gel piece was destained using 50% (v/v) acetonitrile in 100 mm ammonium bicarbonate. After washing with 100 mm ammonium bicarbonate, proteins were reduced with 10 mm dithiothreitol for 1.5 h at 37 °C, and subsequently alkylated with 50 mm acrylamide for 30 min at room temperature. Proteins in the gel were digested with sequencing-grade modified trypsin (Promega, Madison, WI) at 37 °C overnight.