Western blot analysis of P-IRF3(S396) using anti-P-IRF3(S396) antibody (P00165-1).
Electrophoresis was performed on a 10% SDS-PAGE gel at 80V (Stacking gel) / 120V (Resolving gel) for 2 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 C6 whole cell lysates,
Lane 3: mouse brain tissue lysates,
Lane 4: mouse Neuro-2a whole cell 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-P-IRF3(S396) antigen affinity purified polyclonal antibody (P00165-1) 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 1:5000 for 1.5 hour at RT. The signal is developed using an ECL Plus Western Blotting Substrate (Catalog # AR1196-200) with Tanon 5200 system. A specific band was detected for P-IRF3(S396) at approximately 65 kDa. The expected band size for P-IRF3(S396) is at 47 kDa.