Horseradish peroxidase conjugated goat anti rabbit secondary antibody

DRGs, spinal cords, and brains were collected and homogenised in ice-cold lysis buffer containing 50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 5 mM EDTA, 1% Triton X-100, 0.5% Sodium deoxycholate, 0.1% SDS, and standard protease inhibitors. Insoluble material was removed by centrifugation (13,000 r/min × 10 min) and supernatant were collected. Protein concentration for each sample was determined by the bicinchoninic acid method using the MICRO bicinchoninic acid protein assay kit (Pierce). Proteins were loaded on a polyacrylamide gel and separated by electrophoresis. The membrane blots were blocked with 10% non-fat dry milk for 12 h and incubated with primary antibodies: anti-PKG-I (1:100) overnight at 4℃. The membranes were then incubated with horseradish peroxidase-conjugated goat anti-rabbit secondary antibody (1:10000, Amersham Biosciences) for 2 h at room temperature. To normalize the loaded samples, mouse monoclonal anti-tubulin antibody (1:5000; GE Healthcare) was used, followed by incubation with HRP-conjugated goat anti-mouse IgG (1:5000; Pierce). Membranes were incubated with enhanced chemiluminescence reagents (Pierce), and images of the membrane were acquired with the CHEMIL-MAGER chemiluminescence imaging system and analyzed with Image J software. The density of the band of interest was measured and normalized to the density of the band of tubulin.