VLPs were isolated using a similar method to that described elsewhere [35 (link)]. T75 flasks of HEK293T cells were transfected with 30 µg polyethylenimine (PEI) (Sigma, USA), 20 µg of pMExT p67 or pMExT p67HA and co-transfected with 10 µg of either pMEx BLV gag or pTJDNA4. Cells were also transfected with separate plasmids as controls. At 3 days’ post-transfection, media were clarified by centrifugation at 1260× g for 10 min. Supernatants were underlaid with 5 mL 12% (v/v) OptiPrep (Sigma, USA) Tris-buffered saline (TBS) cushions in SS34 tubes and centrifuged at 47,807.6× g for 1 h at 4 °C. Pellets were resuspended in 100 µL ice-cold TBS and placed on glow-discharged carbon-coated copper grids for 30 s. Grids were blocked with 1% (w/v) bovine serum albumin (BSA) diluted in TBS for 1 min, washed thrice with TBS and incubated in rabbit anti-p67 diluted 1:500 in 0.1% (w/v) BSA/TBS for 2 h at 4 °C. These were washed thrice in 1% (w/v) BSA/TBS and incubated with goat anti-rabbit-IgG conjugated to 10 nm colloidal gold (G7402, Sigma, USA) diluted 1:50 in 0.1% (w/v) BSA/TBS for 30 min. Grids were washed thrice with TBS, once with H2O, twice with 2% uranyl acetate and then incubated with 2% uranyl acetate for 1 min. The VLPs were viewed by conventional transmission electron microscopy (TEM) with a Tecnai T20 microscope (FEI, Hillsboro, Oregon, USA).
Free full text: Click here
