Western blot analysis of TNF alpha using anti-TNF alpha antibody (PB9010).
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.
Lane 1: PC-12 Whole Cell Lysate,
Lane 2: Rat Spleen Tissue Lysate,
Lane 3: Rat Brain Tissue Lysate,
Lane 4: Rat Kidney Tissue Lysate,
Lane 5: Rat Liver Tissue Lysate.
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-TNF alpha antigen affinity purified polyclonal antibody (Catalog # PB9010) at 0.5 μg/mL 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 (Catalog # EK1002) with Tanon 5200 system. A specific band was detected for TNF alpha at approximately 26KD. The expected band size for TNF alpha is at 26KD.