Kta avant chromatography system

Two alternative strategies were explored to remove contaminating luciferase proteins from virus stocks. Progeny virions in culture supernatants (IAV stocks) or released through one freeze/thaw cycle from infected cells (RSV stocks) were cleared (4,000×g for 20 minutes at 4°C), then pelleted (60,000×g for 30 minutes at 4°C). Pelleted material was resuspended in TNE buffer (50 mM Tris/Cl pH 7.2, 10 mM EDTA) and purified through a 20/60% one-step sucrose gradient in TNE buffer (100,000×g for 90 minutes at 4°C). Virions were harvested from the gradient intersection. Alternatively, cleared RSV stocks were purified and polished through size exclusion and binding chromatography by passaging through dual functionality Capto Core 700 resin (GE Healthcare) using an ÄKTA avant chromatography system (GE Healthcare). After purification through either method, virus stocks were stored in aliquots at −80°C.