Western blot analysis of Ubiquitin D using anti-Ubiquitin D antibody (M04905-1).
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 Hela whole cell lysates,
Lane 2: human HepG2 whole cell lysates,
Lane 3: human Jurkat whole cell lysates,
Lane 4: human MCF-7 whole cell lysates,
Lane 5: human SH-SY5Y whole cell lysates,
Lane 6: human K562 whole cell lysates,
Lane 7: mouse liver tissue lysates,
Lane 8: mouse HEPA1/6 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-Ubiquitin D antigen affinity purified monoclonal antibody (M04905-1) 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 1:500 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 Ubiquitin D at approximately 23 kDa. The expected band size for Ubiquitin D is at 18 kDa.