Rabbit anti bdnf monoclonal antibody

Extracted protein samples were first quantified using a BCA Protein Assay Kit (Fisher Scientific Co., Pittsburgh, PA, USA) and then boiled in SDS/β‐mercaptoethanol sample buffer. Samples (20 μg) were loaded into each lane of 10% polyacrylamide gels and then the proteins were separated by electrophoresis. Subsequently, the proteins were transferred onto poly(vinylidene difluoride) membranes (Amersham Pharmacia Biotech, St Albans, Herts, UK) by electrophoretic transfer. After 1 h of incubation with 5% BSA, the membrane was incubated with rabbit anti‐BDNF monoclonal antibody (Abcam, Cambridge, MA, USA) or mouse anti‐β‐actin monoclonal antibody (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA) overnight at 4 °C. The specific protein–antibody complex was detected using horseradish peroxidase conjugated goat anti‐rabbit or rabbit anti‐mouse IgG. Detection by the chemiluminescence reaction was carried out using an ECL kit (Pierce, Appleton, WI, USA). The β‐actin signal was used as a loading control.

Western blot analysis was performed as previously described⁴² . Samples were lysed with NP-40 buffer [PBS, 01% TritonX, 05% sodium deoxycholate, 01% sodium dodecyl sulfate (SDS), 50 mM Trix-HCl and 150 mM NaCl, pH 80] containing protease inhibitors (20 mg/ml aprotinin and 1 mM phenylmethylsulfonyl fluoride), 2-mercaptoethanol and 1 mM sodium orthovanadate. Equal concentrations of protein were resolved on 10% SDS-polyacrylamide gels, and then transferred onto Poly-vinylidene Difluoride (PVDF) membranes (ATTO, Tokyo, Japan). The blots were incubated at 4 °C overnight with one of the following primary antibodies: rabbit anti-HIF-1α polyclonal antibody (1:500; Novus Biologicals), rabbit anti–HPRT monoclonal antibody (1:2000; abcam), rabbit anti-BDNF monoclonal antibody (1:500; abcam), mouse anti-β-actin monoclonal antibody (1:10000; Sigma-Aldrich). The blots were subsequently incubated with the appropriate horseradish peroxidase–conjugated secondary antibodies for 90 min and visualized using SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Fisher Scientific, Waltham, MA, USA). The image of each band was captured and analyzed using Image Gauge (Fuji Film, Japan) and ImageJ.