Phosphate buffered saline (pbs)

SDS-PAGE separation of proteins was performed using 12% or 15% (v/v) acrylamide gels following standard procedures [78 (link)]. After separation of ~10 μg protein by SDS-PAGE, proteins were transferred to a nitrocellulose membrane by electro-blotting using 10 mM CAPS [3-(cyclohexylamino)-1-propanesulfonic acid; pH 11], 10% methanol transfer buffer. Membranes were blocked for 1 hr at RT with 50% (v/v) Odyssey blocking buffer in PBS (Licor, USA). Optimal conditions for each rabbit polyclonal antibody were determined empirically and the reproducibility of the results verified by repeated Western blot analyses during these optimisations (data not shown). Membranes were incubated with primary rabbit polyclonal antibodies, in a range from 1:500–1:15000, in blocking buffer containing 0.12–0.25% (v/v) Tween-20 for 1 hr at RT or overnight at 4°C. Following 4 × 5 min washes in 0.1% (v/v) Tween-20, the membranes were incubated in 1:7500 fluorescently labelled goat anti-rabbit IgG secondary antibody (Licor, USA) in blocking buffer containing 0.12–0.25% Tween-20. Membranes were washed again and visualised on a Licor Odyssey imaging system (USA).