Peroxidase conjugated anti rabbit antibodies

Isolated rat brain cerebral cortexes were homogenized and centrifuged at 4 ºC with Triton Lysis Buffer as described earlier (Zielinska et al. 2011 (link)). Total protein concentration in supernatants was determined by the Lowry method using Modified Lowry Protein Assay Reagent (Pierce). Protein (30 μg) was mixed with sample loading buffer, separated on SDS-PAGE and then transferred onto nitrocellulose membrane. The membranes were blocked with 5 % non-fat dry milk in TBS-T buffer. Incubation with antibodies against ASS (1:1,000, Sigma-Aldrich, USA) and ASL (1:1,000, Sigma-Aldrich, USA) was done in TBS-T buffer with 5 % non-fat dry milk at 4 °C temperature over night followed by 10 min incubation with peroxidase-conjugated-anti-rabbit antibodies (1:2,500, Sigma-Aldrich, USA) for detection by SuperSignal West Pico Chemiluminescent Substrate (Pierce). The first antibody was stripped off with 0.1 M glycine, pH 2.9, and second incubation was performed with an antibody against GAPDH (1 h at room temperature), (1:5,000, Sigma-Aldrich, USA).

Approximately 50 mg of rat brain cortex tissue were homogenized in 5 volumes of Triton lysis buffer at 4 °C (20 mM Tris pH 6.8, 137 mM NaCl, 2 mM EDTA, 1 % Triton X-100, 0.5 mM dithiothreitol, and 1 mM phenylmethylsulfonyl fluoride) containing Protease inhibitor cocktail (Sigma-Aldrich, St. Louis, MO), and Phosphatase inhibitor cocktail (Sigma-Aldrich, St. Louis, MO, USA). The brain homogenate was centrifuged for 15 min at 12,000×g, 4 °C. The supernatants were used for total protein determination and Western blot analysis. Briefly, 30 μg of protein was boiled with gel sample buffer (Sigma-Aldrich, St. Louis, MO, USA), separated on sodium dodecyl sulfate–polyacrylamide gel electrophoresis and then transferred onto nitrocellulose membrane. Blots were blocked with 5 % nonfat dry milk in Tris-buffered saline Tween-20 buffer. Incubation with PRMT-1 (A33) antibody (1:1000, Cell Signaling, USA) was done in Tris-buffered saline Tween-20 buffer with 2.5 % nonfat dry milk at room temperature for 0.5 h followed by 10-min incubation with peroxidase-conjugated anti-rabbit antibodies (1:2500, Sigma-Aldrich, St. Louis, MO, USA) for detection by Clarity™ Western ECL Substrate (Bio-Rad, USA). The first antibody was stripped off with 0.1 M glycine and pH 2.9, and the second incubation (1 h, 20–22 °C) was performed with an anti-GAPDH antibody (1:5000, Sigma-Aldrich, Aldrich, St. Louis, MO, USA).