Western blot analysis of HIF-1-alpha/HIF1A using anti-HIF-1-alpha/HIF1A antibody (PB9253).
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 PC-3 whole cell lysates,
Lane 2: human Caco-2 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-HIF-1-alpha/HIF1A antigen affinity purified polyclonal antibody (Catalog # PB9253) 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 HIF-1-alpha/HIF1A at approximately 115-120KD. The expected band size for HIF-1-alpha/HIF1A is at 97KD.