Western blot analysis of Factor VIII using anti-Factor VIII antibody (PA2061).
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: rat kidney tissue lysates,
Lane 2: rat lung tissue lysates,
Lane 3: rat heart tissue lysates,
Lane 4: mouse kidney tissue lysates,
Lane 5: mouse lung tissue lysates,
Lane 6: human Hela whole cell lysates,
Lane 7: human HepG2 whole cell lysates,
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-Factor VIII antigen affinity purified polyclonal antibody (Catalog # PA2061) 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 Factor VIII at approximately 92KD. The expected band size for Factor VIII is at 92KD.