Figure 1. Western blot analysis of MC4R using anti-MC4R antibody (A00619-2). 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 HEK293 whole cell lysates, Lane 3: rat PC-12 whole cell lysates, Lane 4: 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 rabbit anti-MC4R antigen affinity purified polyclonal antibody (Catalog # A00619-2) at 0.5 microg/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 MC4R at approximately 37KD. The expected band size for MC4R is at 37KD.