Novex nupage 4 12 bis tris protein gels

Brain regions (OE, OB, cerebral cortex, cerebellum) were homogenized by a motor-driven pestle on ice in 1× RIPA buffer (Millipore) supplemented with 1 mM phenylmethane sulfonyl fluoride (PMSF), 1 mM Na₃VO₂, 10 mM NaF, phosphatase inhibitor cocktails (Sigma), and Roche Complete Mini EDTA-free Protease Inhibitor Cocktail Tablet (Roche). Homogenates were centrifuged at 10,000 × g for 10 min at 4°C; the supernatants were analyzed by performing Western blotting using 20 μg of total protein on Novex NuPAGE 4–12% Bis-Tris protein gels (Thermo Scientific). Membranes were washed with PBS with 0.05% Tween-20 (PBST), then blocked with 5% milk/PBST or 5% BSA/PBST for 1 hr at 21–23°C, followed by overnight incubation with primary antibodies at 4°C: anti-TNFα (1:250, mouse; R&D Systems), anti-3-nitrotyrosine (1:1,000, rabbit; Millipore), anti-4-hydroxynonenal (1:250, mouse; R&D Systems), anti-olfactory marker protein (OMP) (1:400, goat; Santa Cruz), anti-cleaved caspase 3 (1:1,000, rabbit; Cell Signaling), anti-caspase 3 (1:1,000, rabbit; Cell Signaling), anti-actin (1:10,000, mouse; Sigma). Horseradish peroxidase (HRP; 1:10,000, goat; Jackson)-enhanced chemiluminescence was detected using West Pico Chemiluminescent Substrate (Thermo Scientific). The density of the bands was assessed using ImageJ (Abràmoff et al. 2004 ).