Collagen Type IV

Western blot analysis of Collagen IV using anti- Collagen IV antibody ( Electrophoresis was performed on a 8% 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 HEK293T whole cell lysate, Lane 2: human Hela whole cell lysate, Lane 3: human A549 whole cell lysate. 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-Collagen IV antigen affinity purified monoclonal antibody 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 (ECL1) with Tanon 5200 system. A specific band was detected for Collagen IV at approximately 220KD. The expected band size for Collagen IV is at 161KD.