FIG. 4.

Formation of complexes between CAR fragments and Ad12 knob. (A) Insoluble His-tagged D1 and D1/D2 fragments were refolded, purified, and incubated with a mixture of Ad12 knob (His tag removed by thrombin cleavage) and native Ad2 hexon. CAR fragments were omitted from a control incubation (−CAR). A sample of each mixture was loaded in lanes marked −, and the remainder was then incubated with Ni beads. The beads were washed to remove unbound proteins, and bound material was eluted by boiling in SDS-PAGE sample buffer and loaded into lanes marked +. The positions in the gel of hexon and Ad12 knob (H and K) and the molecular sizes (in kilodaltons) of standards loaded in lane M are indicated. (B) sD1 was partially purified and mixed with purified His-tagged Ad12 knob (lane 1). The mixture was then adsorbed to Ni beads, and a sample of the unbound proteins was loaded in lane 2. Bound proteins were eluted from the beads by boiling in SDS-PAGE sample buffer and were loaded in lane 3. Molecular size standards were loaded in lane M.