Western blot analysis of Sp7/Osterix using anti-Sp7/Osterix antibody (A02077-1).
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 HELA whole cell lysates,
Lane 2: human U87 whole cell lysates,
Lane 3: human MDA-MB453 whole cell lysates,
Lane 4: human MCF-7 whole cell lysates,
Lane 5: human Jurkat whole cell lysates,
Lane 6: human THP-1 whole cell lysates.
Lane 7: rat lung tissue lysates,
Lane 8: rat thymus tissue lysates,
Lane 9: rat skeletal muscle tissue lysates,
Lane 10: rat testis tissue lysates,
Lane 11: mouse lung tissue lysates,
Lane 12: mouse thymus 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-Sp7/Osterix antigen affinity purified polyclonal antibody (Catalog # A02077-1) 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 1:5000 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 Sp7/Osterix at approximately 45KD. The expected band size for Sp7/Osterix is at 45KD.