Horseradish peroxidase hrp conjugated anti mouse secondary antibody

Whole cell protein extracts were harvested with RIPA lysis buffer (P0013B; Beyotime Institute of Biotechnology) consisting of 50 mM Tris (pH 7.4), 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, sodium orthovanadate, sodium fluoride, EDTA and leupeptin. For electrophoresis, 20 µg of each protein extract was loaded onto denatured 12% polyacrylamide gel and transferred to a PVDF membrane (EMD Millipore). The membrane was incubated with 5% skimmed milk for 1 h at room temperature followed by overnight incubation at 4°C with PRX5 (17724-1-AP), NSE (55235-1-AP), SNAP25 (14093-1-AP) and β-actin (60008-1-lg) primary antibodies (all from ProteinTech Group, Inc.). The abovementioned primary antibodies were incubated at a dilution ratio of 1:1,000. For SNAP23 (ProteinTech Group, Inc., 10825-1-AP), the dilution ratio was 1:500. For NeuN detection, the primary antibody was purchased from Cell Signaling Technology, Inc. (no. 12943) and the dilution ratio was 1:1,000. On the following day, the membranes were incubated at room temperature for 1 h with horseradish peroxidase (HRP)-conjugated anti-mouse secondary antibody (ProteinTech Group, Inc., SA00001-1), or HRP-conjugated anti-rabbit secondary antibody (ProteinTech Group, Inc., SA00001-2). HRP signals were detected by enhancing chemical luminol reagent (ProteinTech Group, Inc.) on Amersham Imager 600 (GE Healthcare).