Ti50.2 fixed angle rotor

Honeybee viruses were purified as described previously (13 (link), 16 (link), 52 (link)). Briefly, 50 experimentally infected honeybee pupae were homogenized with a Dounce homogenizer (piston-wall distance of 0.075 mm) in 30 ml of PBS [Dulbecco’s Phosphate-Buffered Saline Modified, D8537, Sigma-Aldrich; 2.7 mM KCl, 136.9 mM NaCl, 1.5 mM KH₂PO₄, and 8.1 mM Na₂HPO₄ (pH 7.4)] on ice. The extract was centrifuged at 15,000g for 30 min at 10°C. The pellet was discarded, and the supernatant was ultracentrifuged at 150,000g for 3 hours in a Ti50.2 fixed-angle rotor (Beckman-Coulter) at 10°C. The resulting pellet was resuspended in PBS in a final volume of 5 ml. MgCl₂ was added to a final concentration of 5 mM, as well as 20 μg/ml of deoxyribonuclease I and 20 μg/ml of RNase. The solution was incubated at room temperature for 30 min and centrifuged for 15 min at 5500g at room temperature. The resulting supernatant was separated using a CsCl (0.6 g/ml) gradient in PBS by ultracentrifugation for 16 hours at 30,000 rpm in an SW41 swinging-bucket rotor at 10°C (Beckman-Coulter). Virus bands were collected by the gentle piercing of ultracentrifuge tubes with an 18-gauge needle. The viruses were buffer exchanged to PBS and concentrated using centrifuge filter units with a 100-kDa molecular mass cutoff.