SDS-PAGE and western blot analysis of RV-VLP gradient fractions.

<p>Electrophoretic separation of selected gradient fractions, which contained structural proteins, out of the 18 fractions of 220 ul collected for each sample is depicted. (<b>I</b>) Gradient fractions for RV-VLPs (dRV-VLP) prepared with baculoviruses confirmed to express VP2 (C2 genotype) and VP6 (I2 genotype). (<b>II</b>) Gradient fractions for RV-VLPs (tRV-VLPs) prepared with baculoviruses confirmed to express VP2 (C2 genotype), VP4 (P<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0105167#pone.0105167-Vesikari1" target="_blank">[4]</a> genotype) and VP6 (I2 genotype). (<b>III</b>) Gradient fractions for RV-VLPs (tRV-VLP) prepared with baculoviruses confirmed to express VP2 (C2 genotype), VP4 (P<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0105167#pone.0105167-Glass1" target="_blank">[8]</a> genotype), VP6 (I2 genotype) and VP7 (G2 genotype). (<b>IV</b>) Detection of VP6 (I2 genotype) and VP7 (G8 genotype) with anti-rotavirus IgG antibodies using western blot in selected gradient fractions in which other structural proteins (VP2, VP5* or VP6) were detected using SDS-PAGE. (<b>V</b>) Gradient fractions for RV-VLPs on SDS-PAGE prepared by step-wise co-infection with baculoviruses confirmed to express VP2 (C2 genotype), VP4 (P<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0105167#pone.0105167-Madhi1" target="_blank">[6]</a> genotype), VP6 (I2 genotype) and VP7 (G2 genotype). (<b>VI</b>) Gradient fractions for RV-VLPs on SDS-PAGE (A) and nitrocellulose membrane, western blot (B) prepared by step-wise co-infection with baculoviruses confirmed to express VP2 (C2 genotype), VP4 (P<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0105167#pone.0105167-Madhi1" target="_blank">[6]</a> genotype), VP6 (I2 genotype) and VP7 (G2 genotype). An approximately 72 kDa non-specific band was consistently present in almost all sucrose gradient fractions #1 to #8 for each sample. The antibody used was weakly reactive against the inner VP2 capsid proteins in the RV-VLPs hence the absence of VP2 band in panel IV. Lane 1, Ladder, PageRuler Plus Prestain Protein Ladder (Fermentas UAB, Vilnius, Lithuania).</p>