Ecl advance blocking agent

Axon or soma protein lysates were boiled in LDS sample buffer and separated on NuPAGE Novex 4–12% Bis–Tris Gels (Life Technologies). Proteins were transferred to Hybond-LFP PVDF membranes (GE Healthcare) and membranes were blocked with ECL Advance Blocking Agent (GE Healthcare) for 1 h, followed by overnight incubation at 4°C with antibodies against TH (Cell Signaling, dilution 1:1000) or β-actin (Cell Signaling, dilution 1:1000). Membranes were washed in 1× TBS-T (1× TBS, 0.1% Tween 20) and incubated with horseradish peroxidase-labeled secondary antibody for 1 h at room temperature. After washing, membranes were developed with ECL Advance Western Blotting Detection Kit reagents, and imaged using Kodak image station 440. Band intensity was quantified using NIH image J Software.

The sensitivity of the antibody 82E1 and the biosimilar Bapineuzumab antibody was further analysed by urea-bicine/bis-tris/Tris/sulphate sodium dodecyl sulphate (SDS)-PAGE followed by western immunoblotting. Stock solutions of synthetic Aβ_1–40 were prepared in sample buffer (0.36 M bis-tris, 0.16 M bicine, 15% wt/vol sucrose, 1% wt/vol SDS and 0.0075% wt/vol bromophenol blue) and stored at −80°C. Different amounts of Aβ_1–40 peptides (25–100 pg and 25–100 ng respectively) were loaded and separated by urea-bicine/bis-tris/Tris/sulphate SDS-PAGE and analysed by immunoblotting as previously described [40 (link)]. The blotting membranes were blocked in 2% enhanced chemiluminescence (ECL) advance blocking agent (GE Healthcare Life Sciences, Little Chalfont, UK) in phosphate-buffered saline (PBS) with 0.075% Tween 20 (PBS-T) for 1h at room temperature and probed with the primary antibody 82E1 or the biosimilar Bapineuzumab antibody (0.5 μg/μl in PBS-T) at 4°C overnight. After three washing steps with PBS-T, biotinylated goat anti-mouse IgG (1:3000 in PBS-T; Life Technologies, Carlsbad, CA, USA) for 82E1 and biotinylated goat anti-human IgG (DAKO, Glostrup, Denmark, 1:3000) for Bapineuzumab, followed by streptavidin-HRP, were employed as secondary reagents. Chemiluminescent signals obtained with ECL Prime Western Blotting Detection Reagent (GE Healthcare Life Sciences) were recorded.

Immunoprecipitated RSK2 and extracts of cells transfected with ZFN and fluorescent protein expression constructs were subjected to SDS-PAGE and the proteins were then transferred to Hybond-ECL nitrocellulose membranes (GE Healthcare). Membranes were blocked with 2% (w/v) ECL Advance Blocking Agent (GE Healthcare), followed by incubation with primary antibodies against active (Ser386-phosphorylated) RSK2 (#AF1094, R&D Systems), total RSK2 (MCA3429Z, ABC Serotec), FLAG epitope tag (horse radish peroxidase-coupled, #F3165, Sigma), EGFP (#8334, Santa Cruz Biotechnology; this antibody was also used to detect Venus and Cerulean) or DsRed2 antibody (#632496, Clontech), as appropriate. Thereafter, relevant horseradish peroxidase-coupled secondary antibodies and ECL Advance (GE Healthcare) were used for visualization of the primary antibodies.