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

Electrophoretic separation of selected gradient fractions, which contained structural proteins, out of the 18 fractions of 220 ul collected for each sample is depicted. (I) Gradient fractions for RV-VLPs (dRV-VLP) prepared with baculoviruses confirmed to express VP2 (C2 genotype) and VP6 (I2 genotype). (II) Gradient fractions for RV-VLPs (tRV-VLPs) prepared with baculoviruses confirmed to express VP2 (C2 genotype), VP4 (P[4] genotype) and VP6 (I2 genotype). (III) Gradient fractions for RV-VLPs (tRV-VLP) prepared with baculoviruses confirmed to express VP2 (C2 genotype), VP4 (P[8] genotype), VP6 (I2 genotype) and VP7 (G2 genotype). (IV) 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. (V) Gradient fractions for RV-VLPs on SDS-PAGE prepared by step-wise co-infection with baculoviruses confirmed to express VP2 (C2 genotype), VP4 (P[6] genotype), VP6 (I2 genotype) and VP7 (G2 genotype). (VI) 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[6] 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).