Collagen Type IV

Western blot analysis of Collagen IV using anti- Collagen IV antibody.Electrophoresis was performed on a 5-20% SDS-PAGE gel at70V (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 placenta 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- Collagen IV antigen affinity purified polyclonal antibody at 0.5 ug/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(ECL) kit ) with Tanon 5200 system. A specific band was detected for Collagen IV at approximately 250KD. The expected band size for Collagen IV is at 161KD.