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 μg/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.

IF analysis of Ubiquitin using anti-Ubiquitin antibody (PB9122).
Ubiquitin was detected in immunocytochemical section of A431 cells. Enzyme antigen retrieval was performed using IHC enzyme antigen retrieval reagent (AR0022) for 15 mins. The cells were blocked with 10% goat serum. And then incubated with 5μg/mL rabbit anti-Ubiquitin Antibody (PB9122) overnight at 4°C. DyLight®488 Conjugated Goat Anti-Rabbit IgG (BA1127) was used as secondary antibody at 1:100 dilution and incubated for 30 minutes at 37°C. The section was counterstained with DAPI. Visualize using a fluorescence microscope and filter sets appropriate for the label used.