Protein analysis of the gradient purified virion preparations.

<p><b>Fig. 6.A.</b> Preparation of RVFV virions grown in C6/36 cells (first passage of Vero E6 virus). <b>6.A.a</b> Silver stained denaturing protein separation electrophoresis gel. <b>6.A.b</b> Immunoblot of the same sample aliquot as in 6.A.a probed with the anti-LGp antibody SW9-22E. <b>6.A.c</b> Immunoblot of the same membrane as in 6.A.b stripped, and re-probed with goat anti-RVFV serum. <b>Fig.6</b><b>.A.d</b> Immunoblot of purified RVFV virions matured in C6/36 cells probed with the antibody SW9-22E. <b>Fig.6</b><b>.A.e</b> Immunoblot of the same membrane as in 6.A.d stripped, and re-probed with goat anti-RVFV serum to illustrate that in independent samples, the monoclonal antibody SW9-E22 recognized only the smaller form of the LGp while the goat RVFV anti-serum detected only the larger form when used for re-probing the membranes. <b>Fig. 6.B.</b> Comparison of the C636 produced virions and the Vero E6 produced RVFV virions. Samples analyzed in Figure 6.A.a and in Figure 6.C.a (lane 1) were analyzed again on the same gel, and probed with anti-LGp antibody SW9-E22. C6 - RVFV virion preparation in C6/36 cells; V6 - RVFV virion preparation in Vero E6 cells. <b>Fig. 6.C.</b> Preparation of RVFV virions grown in Vero E6 cells (fourth passage). This preparation yielded double peak on the sucrose gradient, and the peaks were analyzed separately. Sample 1 are fractions collected at around 58% of sucrose and sample 2 at about 53% of sucrose. <b>6.C.a</b> Silver stained denaturing protein separation electrophoresis gel. Lane designated as 1 is sample 1, lane 2 is sample 2. <b>6.C.b</b> immunoblot of the same sample aliquots as in 6.C.a probed with anti-LGp antibody SW9-22E. <b>6.C.c</b> immunoblot of the same membrane as in 6.C.b stripped, and re-probed with goat anti-RVFV serum. Protein separation gels for immunoblotting had both molecular size markers - colorimetric (Mc) and biotin-labeled (Me), and only the ladder closest to samples of interest is presented. <b>Fig.6</b><b>.D.a</b> Semi-quantification of the gel loading by measuring intensity of the band for the 28 kDa marker, and the band for the N protein on silver stained gels: A.a. protein densities for Fig.6.A.a - virions produced in C6/36 cells in. C.a protein densities for Fig.6.C.a – virions produced in Vero E6 cells. The intensity comparison indicates that the gels with Vero E6 produced virions were overloaded compare to the gels with C6/36 produced virions, considering that equal amounts of the marker proteins were used in all SDS-PAGE. <b>Fig.6</b><b>.D.b</b> Amino acid coverage of the LGp in bold letters as detected by mass spectrophotometry.