Figure. Western blot analysis of TNF alpha using anti-TNF alpha antibody (RP1000).

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: Recombinant Mouse TNFα Protein 0.5ng,

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 # RP1000) 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 17KD. The expected band size for TNF alpha is at 17KD.