VLP purification and quantification

After 24 or 48 hr post-transfection, cell culture supernatants containing Gag-VLPs were collected, filtered through a 0.45 µm filter, and clarified at 800×g for 5 min at 4°C. The supernatant was then purified by loading it on a cushion of 25% sucrose in TNE buffer (25 mM Tris-HCl, 4 mM EDTA, and 150 mM NaCl) and ultracentrifuged at 100,000×g for 1 hr 30 min at 4°C in an SW41Ti rotor (Beckman Coulter). Dry pellets were resuspended in TNE buffer at 4°C overnight. Gag-VLP release was estimated by performing anti-CAp24 immunoblot and by quantifying Gag signal in the blots using Fiji software as described in Thomas et al., 2015. The calculation for Gag-VLP release is: % of Gag in VLP = Gag_released/(Gag_released + Gag_{intracellular normalized to GAPDH}).