Western blot analysis of Claudin 1 using anti-Claudin 1 antibody (M01585).
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 A431 whole cell lysates,
Lane 2: human Caco-2 whole cell lysates,
Lane 3: human A549 whole cell lysates,
Lane 4: human HepG2 whole cell lysates,
Lane 5: rat liver tissue lysates,
Lane 6: 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-Claudin 1 antigen affinity purified monoclonal antibody (Catalog # M01585) at 1:500 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 # EK1002) with Tanon 5200 system. A specific band was detected for Claudin 1 at approximately 23 kDa. The expected band size for Claudin 1 is at 23 kDa.