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. 2011 May 19;6(5):e14813. doi: 10.1371/journal.pone.0014813

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Ye et al.

This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.

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VLPs were produced in Sf9 insect cells using the recombinant baculovirus expression system and purified through a sucrose gradient as described in Materials and Methods. A. Characterization of VLPs by immune-gold-particle labeling and electron microscopy. Purified HA/gp41, HA-C14S/gp41, as well as the control SIV-Gag VLPs were coated onto a sample grid and then incubated with rabbit-anti-HA antibody Jose4 followed by incubation with gold-particle-conjugated goat-anti-rabbit secondary antibodies. The samples were then fixed with 1% uranyl acetate and then examined under an electron microscope. Arrows indicate gold-particle-labeled glycoproteins on the surface of VLPs. B. Detection of HA/gp41 and HA-C14S/gp41 chimeric protein oligomers in VLPs by Western blot. VLPs were lysed with 1% Triton X-100 and then mixed with non-reducing protein sample buffer followed by analysis by SDS-PAGE coupled with Western blot using antibodies against HA (Jose4). Lane M, molecular weight marker; 1, HA-C14S/gp41 VLP, 2, control SIV Gag VLP; 3, HA/gp41 VLP. C. Determining the amount of HA/gp41 and HA-C14S/gp41 chimeric proteins in VLPs by ELISA. VLPs were lysed with 1% Triton X-100 and then coated onto a microtiter plate at 1 µg per well, and the amount of chimeric proteins were detected by ELISA using rabbit-anti-HA antibody Jose4. Control wells were coated with 1 µg of SIV-Gag VLPs. A standard curve was constructed by coating the wells with serial dilutions of purified HA-histag proteins in mixture with 1 µg SIV Gag VLPs and was used for calculating the amount of chimeric proteins in VLPs.