Western immunoblotting was performed on protein A column purified samples containing in vitro synthesized minibodies and in vivo AAV8-derived minibodies. The proteins were separated by SDS-PAGE under reducing or nonreducing conditions and electrophoretically transferred onto a nitrocellulose membrane using the iBLot dry blotting system (Invitrogen). After blocking with 5% skim milk overnight, the blot was probed with an APconjugated human IgG-Fc antibody that was diluted 1:30,000 in blocking buffer for 1 h at room temperature. Excess conjugate was removed by five washes with Phosphate buffered saline containing 0.1% Tween 20 (PBS-T). The detection of protein was performed by incubating the membrane with BCIP/NBT alkaline phosphatase substrate (KPL).