Western Blot Protein Detection

Reduced protein extracts (typically 10–20 µg protein unless stated otherwise) or immunoprecipitates (IPs) were separated on 8% SDS-PAGE gels, or NuPAGE 4–12% Bis-Tris protein gels (Invitrogen) by electrophoresis. Subsequently, proteins were transferred onto polyvinylidene fluoride (PVDF) (Millipore) or nitrocellulose membranes (Amersham), prior to blocking in 5% (w/v) non-fat milk (Marvel) in TBS-T (50 mM Tris–HCl pH 7.5, 150 mM NaCl, 0.2% Tween-20). Membranes were incubated overnight at 4°C in 5% (w/v) bovine serum albumin (BSA)-TBS-T or 5% (w/v) milk-TBS-T with the appropriate primary antibodies. Membranes were subsequently washed in TBS-T and incubated with HRP-conjugated secondary antibodies in 5% (w/v) milk-TBS-T for 1 h at RT. Membranes were subjected to further washing in TBS-T, prior to detection using enhanced chemiluminescence (ECL) reagent (Thermo Scientific) and exposure on medical X-ray films (Konica Minolta) as described previously [12 (link),41 (link)].