Figure 1. Western blot analysis of Anillin using anti-Anillin antibody.
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: human placenta tissue lysate,
Lane 2: human SW620 whole cell lysate,
Lane 3: human A431 whole cell lysate,
Lane 4: human HL-60 whole cell lysate,
Lane 5: human PC-3 whole cell lysate,
Lane 6: mouse NIH3T3 whole 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-Anillin antigen affinity purified polyclonal antibody at 0.5 ug/ml overnight at 4 degree 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 with Tanon 5200 system. A specific band was detected for Anillin at approximately 115KD. The expected band size for Anillin is at 124KD.