Immobilon chemiluminescent substrate

Eosinophils were lysed in 10 mM Tris (pH7.4) containing 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 0.5% CHAPS, and 0.1% NP-40. Samples were separated by reducing SDS-PAGE on 10% Bis-Tris gels (Invitrogen), transferred to nitrocellulose membranes and blocked and probed in 5% milk powder in PBS/0.05% Tween-20. Rabbit antibodies and actin-specific C4 were detected with peroxidase-conjugated secondary antibodies (Jackson) and Immobilon chemiluminescent substrate (Millipore).

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.

The purity of the AAV preparations were confirmed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). For this purpose, the samples were incubated with 6× Laemmle sample buffer (Bio-Rad) with 10% β-mercaptoethanol and boiled for 5 min at 100°C. The denatured proteins were applied to a 10% polyacrylamide gel and run at 120 V. The gel was washed three times with distilled water (diH₂O) and stained with GelCode Blue Protein Safe stain (Invitrogen). In order to confirm and evaluate Rep and Cap expression from the new Rep hybrid plasmids, Western blot analyses were performed. For this purpose, the proteins were transferred to a nitrocellulose membrane following SDS-PAGE by electroblotting. The membrane was blocked in 6% milk in 1× phosphate-buffered saline and probed with hybridoma supernatants containing either monoclonal antibody (MAb) B1, detecting VP1, VP2, and VP3 (47 (link)), or MAb 1F, detecting Rep78, Rep68, Rep52, and Rep40 (30 (link)). After incubation with a secondary antibody with a linked horseradish peroxidase, the proteins were visualized by applying Immobilon chemiluminescent substrate (Millipore) and detection on an X-ray film.

All VLPs analysed by immunoblotting were concentrated by centrifugation through a 20% (w/v) sucrose cushion for 1 hour at 16,000×g, 4°C and resuspended in 1× protein loading buffer. Proteins were separated by SDS polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto polyvinylidene fluoride (PVDF, Millipore) membrane. Immunoblotting was performed with a rat anti-p30^CA (hybrodoma CRL-1912, ATCC), mouse anti-p12 monoclonal (hybrodoma CRL-1890, ATCC), goat anti-p12 polyclonal (a gift from J. Stoye), mouse anti-His (Penta·His Antibody, Qiagen) or rabbit anti-Fv1 (a gift from J. Stoye) followed by anti-rat, anti-goat, anti-mouse or anti-rabbit HRP-conjugated secondary antibodies. Detection was performed using the Immobilon chemiluminescent substrate (Millipore) and hyperfilm processed through a Fijifilm FPM-3800A developer.

Dissected tissue samples were homogenized in RIPA lysis buffer containing 50 mM Tris-HCl, pH 7.4; 150 mM NaCl; 1 mM NaF; 1% NP-40; 0.25% sodium deoxycholate; 1 mM sodium orthovanadate; 1 mM EDTA and a protein protease inhibitor cocktail (Nacalai Tesque, Kyoto, Japan). Lysates containing 10–40 µg of protein were separated by SDS-PAGE and transferred to Hybond ECL Nitrocellulose membranes (GE Healthcare, Buckinghamshire, UK). The membranes were blocked with Blocking One (Nacalai Tesque) for 1 h at room temperature. The membranes were then probed with the following antibodies: rabbit polyclonal anti-Id2 (sc-489; Santa Cruz Biotechnology, Dallas, TX, USA), rabbit monoclonal anti-Id2 (D39E8, #3431; Cell Signaling Technology, Beverly, MA, USA), rabbit polyclonal anti-Mxd1/Mad1 (sc-222; Santa Cruz Biotechnology), rabbit monoclonal anti-c-Myc (D84C12, #5605; Cell Signaling Technology) and mouse monoclonal anti-β-actin (Sigma-Aldrich, Saint Louis, MO, USA) antibodies. After incubation with horseradish peroxidase-conjugated secondary antibodies, bound proteins were detected by incubation with an Immobilon chemiluminescent substrate (Millipore, Billerica, MA, USA).