Western blot analysis of Phospho-MEK1 (S298) using anti-Phospho-MEK1 (S298) antibody (P00292-3).
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 Hela whole cell lysates,
Lane 2: human A431 whole cell lysates,
Lane 3: human U251 whole cell lysates,
Lane 4: human SIHA whole cell lysates,
Lane 5: rat lung tissue lysates,
Lane 6: mouse lung 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-Phospho-MEK1 (S298) antigen affinity purified monoclonal antibody (P00292-3) at 1:1000 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 1:500 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 Phospho-MEK1 (S298) at approximately 54 kDa. The expected band size for Phospho-MEK1 (S298) is at 43 kDa.