Western blot analysis of Optineurin/OPTN using anti-Optineurin/OPTN antibody (M00952).
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 U-87MG whole cell lysates;
Lane 2: human U20S whole cell lysates;
Lane 3: human placenta tissue lysates;
Lane 4: human Hela whole cell lysates;
Lane 5: human A549 whole cell lysates;
Lane 6: human K562 whole cell lysates;
Lane 7: rat C6 whole cell lysates;
Lane 8: mouse Neuro-2a whole cell 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 mouse anti-Optineurin/OPTN antigen affinity purified monoclonal antibody (Catalog # M00952) 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-mouse 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 # EK1001) with Tanon 5200 system. A specific band was detected for Optineurin/OPTN at approximately 66KD. The expected band size for Optineurin/OPTN is at 66KD.