Western blot analysis of GAD67 using anti-GAD67 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: Rat Brain Tissue Lysate, Lane 2: Rat Testis Tissue Lysate, Lane 3: MCF-7 Whole Cell Lysate, Lane 4: MM231 Whole Cell Lysate, Lane 5: HELA Whole Cell Lysate, Lane 6: SMMC Whole Cell Lysate, Lane 7: COLO320 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-GAD67 antigen affinity purified polyclonal antibody 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 with Tanon 5200 system. A specific band was detected for GAD67 at approximately 67KD. The expected band size for GAD67 is at 67KD.