Western blot analysis of TIMP3 using anti-TIMP3 antibody (PB9831).
Electrophoresis was performed on a 5-20% SDS-PAGE gel at 70V (Stacking gel) / 90V (Resolving gel) for 2-3 hours.
Lane 1: Rat Kidney Tissue Lysate at 50ug,
Lane 2: Mouse Ovary Tissue Lysate at 50ug,
Lane 3: HELA Whole Cell Lysate at 40ug,
Lane 4: MCF-7 Whole Cell Lysate at 40ug,
Lane 5: SMMC Whole Cell Lysate at 40ug.
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-TIMP3 antigen affinity purified polyclonal antibody (Catalog # PB9831) 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 TIMP3 at approximately 34 kDa. The expected band size for TIMP3 is at 34 kDa.