Western blot analysis of Cytochrome P450 2E1/CYP2E1 using anti-Cytochrome P450 2E1/CYP2E1 antibody (M00672-2).
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 30 ug of sample under reducing conditions.
Lane 1: human HCCP tissue lysates,
Lane 2: rat liver tissue lysates,
Lane 3: mouse liver tissue lysates.
After electrophoresis, proteins were transferred to a nitrocellulose membrane at 150 mA for 50-90 minutes. Blocked the membrane with 5% non-fat milk/TBS for 1.5 hour at RT. The membrane was incubated with mouse anti-Cytochrome P450 2E1/CYP2E1 antigen affinity purified monoclonal antibody (Catalog # M00672-2) 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-mouse 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 # EK1001) with Tanon 5200 system. A specific band was detected for Cytochrome P450 2E1/CYP2E1 at approximately 56 kDa. The expected band size for Cytochrome P450 2E1/CYP2E1 is at 56 kDa.