Proteins were blotted onto polyvinylidene fluoride membranes for 1 h at a constant current of 2 mA/cm2 in a Transblot semidry transfer cell (Bio-Rad) followed by two washes in Tris buffered saline (TBS buffer, 20 mM Tris-HCl, pH 7.5, 150 mM sodium chloride) for 10 min each at room temperature, blocking of the membrane at 4C overnight in TBS buffer containing 10% skim milk powder (block buffer), three washes at room temperature in TBS-Tween/Triton buffer (TBS buffer, 0.05% (v/v) Tween 20, 0.2% (v/v) Triton X-100) and incubation with the primary antibody (equal mix of anti-185/333 antisera diluted 1:10000 in block buffer). This was followed by three washes in TBS-Tween/Triton buffer for 10 min each at room temperature and incubation in the secondary antibody (goat anti rabbit IgG Alkaline Phosphatase conjugate, 1:20000 in block buffer) and three final washes in TBS-Tween/Triton buffer. Protein-antibody complexes were visualised by colour reaction with 3 ml BCIP/NBT per 150 cm2 membrane area for 2-5 min at room temperature and photographed using a gel documentation system.

Sham Nair 2014