Western blot analysis of FADD using anti-FADD antibody (PA1464).
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 30 ug of sample under reducing conditions.
Lane 1: human HT1080 whole cell lysates,
Lane 2: human A549 whole cell lysates,
Lane 3: human Jurkat whole cell lysates,
Lane 4: human A431 whole cell lysates,
Lane 5: human HepG2 whole cell lysates,
Lane 6: human Hela whole cell lysates,
Lane 7: human MCF-7 whole cell lysates,
Lane 8: human Raji whole cell lysates.
After electrophoresis, proteins were transferred to a nitrocellulose membrane at 150 mA 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-FADD antigen affinity purified polyclonal antibody (Catalog # PA1464) 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 1:5000 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 FADD at approximately 28 kDa. The expected band size for FADD is at 23 kDa.