Figure 4

Sucrose gradient analysis of L1 VLPs and capsomere derivatives. Ultracentifugation with 5–20% sucrose gradient in PBS/1 M NaCl was used to resolve purified L1 VLPs (top panel), 3-1 (middle panel), and 423-3 (lower panel). For each fraction, 12 μl was resolved on 10%/5% SDS-PAGE, transferred onto nitrocellulose, and visualized using CAMVIR-1 anti-L1 mAb (1:60,000 dilution), goat anti-mouse IgG-HRP conjugate (1:20,000 dilution) and chemiluminescence. Molecular weights are shown to the left of the standard ladder. For each fractionation, sedimentation standards were concurrently resolved (bovine serum albumin, 4.6S; bovine catalase, 11.3S, and E. coli β-galactosidase, 19S). For the first two standards, the peak fractions for each gradient are shown on top of the panel; under our typical ultracentrifugation parameters (16–20 hours) and buffer composition with PBS/1 M NaCl as the sucrose solvent, we consistently observed that the 19S standard migrated to the bottom of the tube into the pellet (P) fraction. Note that the VLPs are found in the pellet fraction while the capsomere preparations were resolved across the gradient (see Results). Note also that there is an 80 kD band that is enriched around the 4.6S standard-containing fractions in all of our sucrose gradient-derived immunoblots (this Figure and data not shown). Since this band was not seen in immunoblots of pre-gradient L1 preparations, we assume that this represents an artifact from one or more of the sedimentation standards.