Figure 1. Western blot analysis of Afm using anti-Afm antibody (A00275-3). 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 heart tissue lysates, Lane 2: rat liver tissue lysates, Lane 3: rat kidney tissue lysates, Lane 4: rat spleen tissue lysates, Lane 5: mouse spleen tissue lysates, Lane 6: mouse thymus tissue lysates, Lane 7: mouse ANA-1 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-Afm antigen affinity purified polyclonal antibody (Catalog # A00275-3) at 0.25 microg/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 Afm at approximately 87KD. The expected band size for Afm is at 87KD.