Figure 1. Western blot analysis of CD86 using anti-CD86 antibody (A00220-1). 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 spleen tissue lysate,. Lane 2: rat liver tissue lysate,. Lane 3: rat PC-12 cell lysate,. Lane 4: mouse spleen tissue lysate,. Lane 5: mouse thymus tissue lysate,. Lane 6: mouse brain tissue lysate,. Lane 7: human COLO-320 cell lysate. 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-CD86 antigen affinity purified polyclonal antibody (Catalog # A00220-1) at 0.5 ug/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 CD86 at approximately 65KD. The expected band size for CD86 is at 38KD.