SDS-PAGE Analysis of rAAV Purity

The purity of the rAAV serotype was examined by SDS-PAGE. rAAV samples with 1010 viral genomes were boiled in the 5 x oading buffer (100 mM Tris-HCl, pH 6.8, 4% SDS, 200 mM DTT, 0.2% bromophenol blue, and 20% glycerol) for 5 min, and loaded on a 10% polyacrylamide gel. The samples were separated by SDS-PAGE and analyzed via sequential Western blotting as described previously [70 (link),73 (link)]. Briefly, the samples were transferred to the nitrocellulose membrane (Amersham Protran 0.45 µm Membrane; Cytiva, MA, USA). The membrane was blocked with 5% non-fat milk in Tris-buffered saline containing 0.05% Tween (TBST) and incubated with the primary antibody overnight. The membrane was washed thrice in TBST and incubated with the secondary horseradish peroxidase (HRP)-conjugated antibody (Santa Cruz, 1:3000) for one hour. Immunoreactive proteins were further detected by the ECL Western blotting substrate (Gibco, MD).