SDS-PAGE analysis, with Coomassie blue staining, of virus generation tests. The negative control is marked Ø. Briefly, a pFastBac1 construction was used to transform E. coli strain DH10Bac to produce recombinant Bacmids. Purified recombinant Bacmids were then prepared and transfected in Spodoptera frugiperda (Sf) cells to generate P1 virus stock. The cells were collected by centrifugation. The supernatant (medium) was lysed in a buffer containing PBS, pH 7.5. After centrifugation, the soluble supernatnant fraction was collected (native protein extraction - NPE). The insoluble fraction was collected and solubilized with a denaturing buffer. After centrifugation, the supernatant was collected (denatured protein extraction - DPE). Two different clones, 1 and 2, were tested. The medium, NPE and DPE fractions were analyzed by SDS-PAGE.
Western blot (ECL) analysis, with His-tag antibody, of virus generation tests. Based on the virus generation tests, expression tests were carried out with the P1 stock obtained with Bacmid clone 1.
SDS-PAGE analysis, with Coomassie blue staining, of expression and amplification tests. The negative control is marked Ø. Briefly, Sf cells were infected with P1 virus stock to obtain the P2 virus stock. Sf cells and Trichoplusia ni (Tn) cells were infected with different quantities of P2 stock to determine the optimal multiplicity of infection (MOI) and incubation time.
Western blot (ECL) analysis, with His-tag antibody, of expression and amplification tests. Based on these tests, the optimal expression was observed in Tn cells incubated with 30 µl of virus (MOI approx. 1) for 48 h (D2).
A) SDS-PAGE analysis, with Coomassie blue staining, of YFVE Protein after purification. Briefly, the equilibration buffer used was PBS, pH 7.5. There were 2 wash steps, using PBS, pH 7.5, with increasing concentration of imidazole with each wash (30 and 50 mM respectively). The elution buffer was PBS, pH 7.5, 300 mM imidazole. B) Western blot analysis of YFVE Protein after purification.
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