Western Blot Protein Detection Protocol

Proteins were resolved by Bis–Tris SDS-polyacrylamide gel electrophoresis (PAGE) and then transferred onto polyvinylidene difluoride membranes using wet or semi-dry immunoblotting. Membranes were blocked with 5% nonfat dry milk or BSA in TBS-T (0.1% TWEEN-20) typically for 1 h, and then probed with primary antibody in 5 % nonfat dry milk or BSA (in TBS-T) at the indicated dilutions (as described below) overnight at 4°C. Membranes were washed in TBS-T and probed with HRP-linked anti-mouse or anti-rabbit antibody in 5% nonfat dry milk or BSA (in TBS-T) for 1 h at room temperature. Proteins were visualized using an enhanced chemiluminescence reagent (Western Lightning Plus-ECL, PerkinElmer) or the SuperSignal West Pico PLUS or Femto chemiluminescence reagents (Thermo Fisher Scientific) on an Amersham Imager 600 (General Electric) or ImageQuant LAS 4000 Chemiluminescent Image Analyzer (General Electric) as previously described (53 (link)). Primary and secondary antibodies for Western blotting are listed below.