Western Blot Protein Detection Protocol

Total cell protein extracts were prepared using RIPA buffer as previously described [26 (link)]. All protein samples were electrophoresed in a SDS-polyacrylamide gel and transferred onto PVDF membranes. PVDF membranes were blocked for 2 hours with 5% non-fat milk or 5% BSA in TBS containing 0.1% Tween-20, and then incubated overnight at 4°C with primary antibodies. Membranes were washed three times with TBS-Tween 0.05% and incubated with HRP-conjugated anti-rabbit IgG or anti-mouse IgG secondary antibodies for 1 hour at room temperature in 5% non-fat dry milk in TBS-Tween. Immunoreactive bands were developed with Lumi-light^PLUS Western Blotting Kit (Roche, Molecular Biochemicals) and visualized using X-ray films (Amersham Hyperfilm ECL, GE Healthcare, Buckinghamshire, UK).