Fig 1: Comparative characterizations of N1-based VLPs with their prototyped H5N1-VLP.(A) Secreted VLPs of different designs are as indicated to the left of panels. Purified VLPs were adsorbed onto formvar/carbon-coated nickel grids. NS, negatively stained with 2% uranyl acetate; immunogold stained with specific antibodies as marked on the top. The secondary antibodies were goat anti-rabbit or anti-mouse conjugated to 12 nm gold beads. The grids were observed by TEM at 100,000× magnification. (B) Western blot analysis of virus-specific proteins in VLPs. Equal amount of VLPs were separated on a 7.5–12.5% gradient gel followed by Coomassie blue staining or western blot analysis with specific antibodies. Identity of viral proteins in the VLPs were detected and labeled on the right. The same blot was probed with anti-tubulin antibody as a loading control of VLP preparations. Middle, the HA and NA amount in 0.5 µg of each VLP was marked below. (C) Assessment of HA function by hemagglutination assay. The amounts of VLPs used are indicated, in a two-fold serial dilution. TNE (buffer of VLPs) was used as the negative control. The antibodies used in this study were tubulin (ab6160), N1 (ab21305), and M2e (ab5416) from Abcam (Cambridge, MA). Rabbit polyclonal antibody against H5 was provided by Dr. Che Ma (Genomics Research Center, Academia Sinica).