Western blot analysis of ADO using anti-ADO antibody (PB10032). 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 brain tissue lysates, Lane 2: rat testicular tissue lysates, Lane 3: rat spleen tissue lysates, Lane 4: human MCF-7 whole cell lysates, Lane 5: human SW620 whole cell lysates, Lane 6: mouse spleen tissue lysates, Lane 7: mouse heart tissue lysates, Lane 8: mouse HEPA1-6 whole cell lysates, Lane 9: mouse SP2/0 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-ADO antigen affinity purified polyclonal antibody (Catalog # PB10032) 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 ADO at approximately 28,30KD. The expected band size for ADO is at 30KD.