Western blot analysis of Cyclin D1 using anti-Cyclin D1 antibody (PA1245-2). 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 Testis Tissue Lysate, Lane 2: Human Placenta Tissue Lysate, Lane 3: Rat Brain Tissue Lysate, Lane 4: MCF-7 Whole Cell Lysate, Lane 5: COLO320 Whole Cell Lysate, Lane 6: SW620 Whole Cell Lysate, Lane 7: MM231 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-Cyclin D1 antigen affinity purified polyclonal antibody (Catalog # PA1245-2) 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 Cyclin D1 at approximately 33KD. The expected band size for Cyclin D1 is at 33KD.