Nupage novex precast 4 to 12 bis tris gradient gels

Cells were washed with PBS and lysed by use of RIPA buffer containing a protease inhibitor cocktail (Sigma Life Science and Biochemicals, St. Louis, MO). For HBV protein preparation, samples were heated at 56°C for 30 min. We loaded equal quantities of protein (20 μg) in all lanes. Protein samples were separated by SDS-PAGE with NuPAGE Novex precast 4 to 12% Bis-Tris gradient gels (Invitrogen, Carlsbad, CA) and blotted onto nitrocellulose membranes. The membranes were blocked with 5% bovine serum albumin (BSA) in Tris-buffered saline with Tween 20 (TBST). Primary antibodies included mouse anti-human PKLR (Santa Cruz Biotechnology, TX), mouse anti-HCV core (Fisher Scientific, Pittsburgh, PA), mouse anti-HBcAg (Abcam, Cambridge, MA), and mouse anti-β-actin (Sigma, St. Louis, MO). The secondary antibodies were horseradish peroxidase (HRP)-conjugated enhanced chemiluminescence (ECL) donkey anti-rabbit IgG and HRP-conjugated ECL sheep anti-mouse IgG (GE Healthcare Biosciences, Pittsburgh, PA). The blots were subjected to chemiluminescence assay by use of an Amersham ECL Western blotting detection kit (GE Healthcare Biosciences, Pittsburgh, PA).