Purification of HEK-293T/17 generated LASV VLP by sucrose gradient sedimentation and detection of GP1, GP2, NP, and Z proteins in fractions by western blot analysis. LASV VLP were precipitated with PEG-6000/NaCl and concentrated by ultracentrifugation. Pellets were resuspended in 500 μL of TNE or PBS, overlayed on discontinuous 20 - 60% sucrose gradients, and sedimented by ultracentrifugation. Eight fractions of 500 μL each were collected from sucrose gradients. Ten μL from each fraction were separated on denaturing 10% NuPAGE gels, blotted and probed with LASV protein-specific mAbs. LASV VLP packaging Z+GPC+NP (A) and Z+GPC (B) were analyzed for distribution of GP1 (Ai, Bi), GP2 (Aii), NP (Aiii), and Z (Aiv, Bii) throughout the gradient spectrum. Fraction 1 contained input supernatant (S) loaded onto gradients. Fractions 2 through 8 were from 20 - 60% sucrose gradients. Lane 9 contained insoluble material that pelleted through 60% sucrose (P). The size of each protein in kDa is indicated to the right of each blot (unprocessed GPC: 75 kDa, GP1: 42 kDa, GP2: 38 kDa, NP: 60 kDa, and Z: 12 kDa).
Branco et al. Virology Journal 2010 7:279 doi:10.1186/1743-422X-7-279