Horseradish peroxidase hrp linked anti mouse igg antibody

rAAV-GFP samples were diluted to 2 × 10¹⁰ vector genome containing particles/mL (~2 ng/μL) in citrate-phosphate with 150 mM NaCl at pH 7.4, 6.0, 5.5, or 4.0. Samples were then heated in a Bio-Rad C1000 thermo cycler for 5 min and cooled to 4 °C. After cooling, 10 μL of the heated samples were loaded onto a nitrocellulose membrane by vacuum suction using a Minifold Dot-Blot apparatus (GE Healthcare). Membranes were blocked in 5% milk/Tween-PBS for 1 h at RT or overnight at 4 °C. Membranes were probed for intact capsids using ADK1a (AAV1) [29 (link)], A20 (AAV2) (ARP, Waltham, MA, USA) [30 (link)]), ADK5a (AAV5) [29 (link)], and ADK8 (AAV8) [31 (link)]; denatured capsids using B1 [30 (link)]; and the presence of VP1u using A1 (ARP) [30 (link)] in 1% milk/Tween-PBS for 1 h at RT. Unbound antibodies were removed by three washes in Tween-PBS (5 min each), after which the membrane was incubated with a secondary horseradish peroxidase (HRP)-linked anti-mouse IgG antibody (GE Healthcare). After 1 h incubation, the membrane was washed three times in Tween-PBS to remove unbound antibodies (5 min each). The membrane was incubated with Immobilon chemiluminescent substrate (Millipore) and imaged to detect the presence of intact capsids, denatured capsids, or VP1u.

Dulbecco’s Modified Eagle’s medium (DMEM), phosphate-buffered saline (PBS), and Hanks’ balanced salt solution were obtained from Nissui (Tokyo, Japan). Fetal bovine serum (FBS) and calf serum (CS) were obtained from JRH Bioscience (Tokyo, Japan). Anti-Myc monoclonal antibody (9E10) was purchased from Millipore (Billerica, MA, USA). Anti-FLAG M2 antibody and anti-α-tubulin antibody (B-5-1-2) were obtained from Sigma-Aldrich (St. Louis, MO, USA). Anti-GFP monoclonal antibody (B-2) was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-PI3KAP/XB130 antibody was raised in our laboratory as previously described (12 (link)). Alexa Fluor 488-conjugated anti-mouse IgG antibody was from Invitrogen (Carlsbad, CA, USA). Horseradish peroxidase (HRP)-linked anti-mouse IgG antibody and HRP-linked anti-rabbit IgG antibody were purchased from GE Healthcare (Buckinghamshire, UK). Other chemicals were of reagent grade available commercially.