FIG 2.
Overlay assay using recombinant GN and F. occidentalis first-instar proteins resolved in two-dimensional gels. Total proteins (150 μg) extracted from pooled healthy first-instar larvae (0 to 17 h old) of F. occidentalis were resolved by 2D gel electrophoresis and transferred to nitrocellulose membranes. The membranes were incubated overnight with blocking buffer (negative control) (A) or recombinant TSWV GN (3.5 μg/ml) (B). Using the polyclonal rabbit anti-TSWV GN, protein spots that consistently bound to the recombinant TSWV GN in two (spots 1 through 11) biological replicates of the overlay assay were collected from two 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.