Western blot analysis of 4EBP1/EIF4EBP1/2/3(Phospho-T46/T46/T32) using anti-4EBP1/EIF4EBP1/2/3(Phospho-T46/T46/T32) antibody (M00968T46T32).
Electrophoresis was performed on a 12% 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: human HEL whole cell lysates,
Lane 2: human SH-SY5Y whole cell lysates,
Lane 3: human HepG2 whole cell lysates,
Lane 4: human MCF-7 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-4EBP1/EIF4EBP1/2/3(Phospho-T46/T46/T32) antigen affinity purified monoclonal antibody (M00968T46T32) 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-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 4EBP1/EIF4EBP1/2/3(Phospho-T46/T46/T32) at approximately 18 kDa. The expected band size for 4EBP1/EIF4EBP1/2/3(Phospho-T46/T46/T32) is at 18 kDa.