Immunoblotting Protein Detection Protocol

Immunoblotting was performed essentially as described elsewhere (Eyre et al, 2010 (link)). Briefly, membrane-bound protein was blocked with 5% skim milk in 0.1% TBS-T for 1 h and then incubated in the appropriate dilution of primary antibody in 1% skim milk overnight at 4°C. Thereafter, membranes were incubated with horseradish peroxidase-conjugated secondary antibody for 1 h and washed before detection using either the ECL Plus Western blotting detection reagent kit (Amersham Pharmacia Biotech) or the Supersignal West Femto Maximum Sensitivity Substrate detection kit (Thermo Fisher Scientific) as per the manufacturer’s instructions. Protein bands were visualized by a Chemi DocTM MP Imaging System (Bio-Rad).