Iblot gel transfer stack nitrocellulose

Purified rAAV vectors were run on SDS-PAGE Bis-Tris 4–12% gels (Nu-PAGE, Invitrogen), and either directly Coomassie stained or transferred to iBlot gel transfer stack nitrocellulose (Invitrogen) prior to immuno detection. Mouse IgG1 (derived from clone B1 recognizing a C-terminal region; Progen) was used in a 1/250 dilution as antibody against AAV VP of the studied serotypes. The Anti Rep mouse IgG1 clone 303.9 (Progen) was used in a 1/100 dilution. Anti VP polyclonal antibody VP51 (Progen) was used in a 1/250 dilution. The secondary antibody was a goat anti-mouse Dye 680 (LI-COR) used in a 1/5000 dilution. Antibody incubations were performed in Odyssey blocking buffer (LI-COR) and colour visualisation was performed on the Odyssey system (LI-COR).

Purified rAAV vectors were run on SDS-PAGE Bis-Tris 4–12% gels (Nu-PAGE, Invitrogen, Waltham, MA, USA) and either directly Coomassie-stained or transferred on nitrocellulose membrane (iBlot gel transfer stack nitrocellulose, Invitrogen, Waltham, MA, USA) prior to Western blot immunodetection. Anti-VP primary antibody mouse igG1 clone B1 (Progen) was used at a 1/250th dilution. The secondary antibody was goat anti-mouse dye 680 (LI-COR) used at a 1/5000th dilution. Hybridization was performed in infrared imaging system blocking buffer (LI-COR), and revelation was performed on an Odyssey system (LI-COR).