Western blot analysis of ClC-5/CLCN5 using anti-ClC-5/CLCN5 antibody (A02286-2).
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: rat brain tissue lysates,
Lane 2: rat liver tissue lysates,
Lane 3: rat heart tissue lysates,
Lane 4: rat kidney tissue lysates,
Lane 5: mouse brain tissue lysates,
Lane 6: mouse liver tissue lysates,
Lane 7: mouse heart tissue lysates,
Lane 8: mouse kidney 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 rabbit anti-ClC-5/CLCN5 antigen affinity purified polyclonal antibody (Catalog # A02286-2) 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 1:5000 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 ClC-5/CLCN5 at approximately 100 kDa. The expected band size for ClC-5/CLCN5 is at 91 kDa.