FIG. 1.

Cy3 conjugation to AAV capsids. HeLa cells were infected with rAAV labeled with Cy3 as explained in Materials and Methods. (A) Fluorescent colocalization of Cy3AAV (left panel in red) with AAV capsid proteins stained with FITC-labeled anti-cap B1 antibody (center panel in green). The two channels are superimposed in the right panel, demonstrating colocalization of Cy3AAV and capsid proteins (in yellow). SDS-PAGE analysis, following staining with Sypro-Orange, was used to evaluate the purity of the rAAV preparation used for Cy3 labeling (B). Lane 1 contains molecular size markers, and the sizes (in kilodaltons) are given to the left of the gel. Various amounts of rAAV from 20 to 1 μl were loaded onto lanes 2 to 7 in a 12% polyacrylamide–Tris–glycine gel (Novex). The locations of capsid proteins VP-1, -2, and -3 are marked to the right of the gel. Lane 8 contains 50 ng of β-galactosidase protein as a reference. (C) Ultrastructure of unlabeled (upper panel) and Cy3-conjugated AAV (lower panel) by transmission electron microscopy. The binding of Cy3-labeled AAV to its receptor on HeLa cells was tested for competition by increasing amounts of heparin (D). Numbers below each panel indicate the doses of heparin used in these assays.