Western blot analysis of NGFR expression in (1) C6 cell lysate; (2) PC-12 cell lysate. Electrophoresis was performed on a 5-20% SDS-PAGE gel at 70V (Stacking gel) / 90V (Resolving gel) for 2-3 hours. The sample well of each lane was loaded with 50ug of sample under reducing conditions. After Electrophoresis, proteins were transferred to a Nitrocellulose membrane at 150mA for 50-90 minutes. Blocked the membrane with 5% Non-fat Milk/ TBS for 1.5 hour at RT. The membrane was incubated with rabbit anti- NGFR monoclonal antibody (Catalog # M01187-1) overnight at 4°C, then washed with TBS-0.1%Tween 3 times with 5 minutes each and probed with a goat anti-rabbit IgG-HRP secondary antibody at a dilution of 1:10000 for 1.5 hour at RT. The signal is developed using an Enhanced Chemiluminescent detection (ECL) kit with Tanon 5200 system.
A specific band was detected for NGFR

Immunohistochemical analysis of paraffin-embedded human glioma, using NGFR Antibody NGFR was detected in paraffin-embedded tissue section. Heat mediated antigen retrieval was performed in citrate buffer (pH6, epitope retrieval solution) for 20 mins. The tissue section was blocked with 10% goat serum. The tissue section was then incubated with 1ug/ml rabbit anti-NGFR Antibody overnight at 4°C. Biotinylated goat antirabbit IgG was used as secondary antibody and incubated
for 30 minutes at 37°C. The tissue section was developed using Strepavidin-Biotin-Complex (SABC)with DAB as the chromogen.