FIG 1.
Overlay assay using purified virions and F. occidentalis first-instar proteins resolved in two-dimensional gels. Total proteins (150 μg) extracted from pooled noninfected first-instar larvae (0 to 17 h old) of F. occidentalis were resolved by 2D gel electrophoresis and transferred to nitrocellulose membranes. After blocking, membranes were incubated overnight with blocking buffer (negative control) (A) or purified TSWV at 25 μg/ml (B), followed by incubation with polyclonal rabbit anti-TSWV GN antibodies. Only protein spots that consistently bound to purified TSWV in three (spots 1, 2, 4, 6, and 7) and four (spots 3, 5, and 8) biological replicates of the overlay assay were collected from three individual picking gels and subjected to ESI-mass spectrometry for protein identification. Protein spots observed in the no-overlay-control membrane represent nonspecific binding and were not collected for further analysis. Molecular mass (in kilodaltons) is shown on the y axis, and pI (as pH range) is shown on the x axis.