Figure 1. Western blot analysis of Ubiquitin using anti-Ubiquitin antibody (PB9122). 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 50ug of sample under reducing conditions. Lane 1: human Caco-2 whole cell lysates, Lane 2: human K562 whole cell lysates, Lane 3: human THP-1 whole cell lysates, Lane 4: rat PC-12 whole cell lysates, Lane 5: rat brain tissue lysates, Lane 6: mouse brain tissue lysates, After Electrophoresis, proteins were transferred to a Nitrocellulose membrane at 150mA 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 antigen affinity purified polyclonal antibody (Catalog # PB9122) at 0.5 microg/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 1:10000 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 Ubiquitin at approximately 9KD. The expected band size for Ubiquitin is at 9KD.