Avb sepharose hi trap column

All vectors were prepared by a triple transfection method as previously described in HEK293 cells (Howard et al., 2008 (link)). Cell and media pellets were thawed and combined, then freeze-thawed two times, vortexing after each thaw. MgCl₂ (2 mM final; Sigma-Aldrich, St. Louis, MO) and Benzonase (EMD Millipore, Billerica, MA) were added at 50 U/ml of cell solution for 1 h at 37°C with continuous shaking. The solution was centrifuged for 20 min at 2450 × g at 4°C. The supernatant was transferred to 75 ml of phosphate-buffered saline (PBS) with 2 mM MgCl₂ and sequentially filtered through 5-, 0.45-, and 0.22-μm filters. The solution was then run through a 1 ml of AVB Sepharose HI-TRAP column (GE Healthcare, Pittsburgh, PA) using an AKTA purifier (GE Healthcare) at a rate of 2 ml/min and eluted using a 15 mM sodium citrate solution (Sigma-Aldrich) at a rate of 1 ml/min. The fractions containing the peak of the OD₂₅₄ and OD₂₈₀ readings were collected and dialyzed using a 10,000 MWCO dialysis cassette (Pierce, now Thermo Fisher Scientific, Rockford, IL) in 1 l of PBS containing 0.5 mM MgCl₂ for three exchanges over 25–30 h. The equilibrated virus was aliquoted, snap-frozen, and stored at −70°C.