FIG. 5.

Western blot analysis of wt and mutant AN. AN was purified as described in Materials and Methods. Samples include His-tagged rAN (lane 1) and the mutants indicated (lanes 2 and 3). Controls show that there is no material binding to the affinity resin from extracts of uninfected Sf-9 cells (lane 4) or wt AcMNPV-infected cells (lane 5). For these control assays (lanes 4 and 5), the cells were carried through the purification protocol, fractions E6 and E7 (Fig. 2) were pooled, and about three times the volume used for the His-tagged enzyme-containing extracts (about 24 μl) was loaded onto the gel. Lanes 6 and 7 contain 10 μl of dialysate from cells infected with wt AcMNPV and recombinant AcMNPV expressing AN, respectively. Samples were electrophoresed through sodium dodecyl sulfate–10% polyacrylamide gels (20) and electroblotted onto polyvinylidene difluoride membranes (Micron Separations, Inc.) for 2 h at 185 mA; then, Western blot analyses were carried out as previously described (27). Samples were treated with 1:1,000 dilution of the antiserum, and the second antibody (goat anti-rabbit conjugated to horseradish peroxidase; Promega) was used at 1:2,500. The positions of selected size standards are shown in kilodaltons on the left. The estimated values for the major immunoreactive bands are shown on the right.