TTL Antibody / Tubulin-tyrosine ligase (FY13081)

Western blot analysis of TTL using anti-TTL antibody. Electrophoresis was performed on a 10% SDS-PAGE gel at 80V (Stacking gel) / 120V (Resolving gel) for 2 hours. Lane 1: rat brain tissue lysates, Lane 2: mouse brain tissue lysates. 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-TTL antibody at 0.5 ug/ml overnight at 4oC, 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 was developed using an ECL Plus Western Blotting Substrate. A dominant band is detected at ~43 kDa, consistent with the predicted size of TTL. Two faint bands flanking the main band are observed and likely reflect differential phosphorylation (slower) and/or limited proteolysis (faster). Additional weak bands appear as a ~55 kDa doublet and a ~100 kDa doublet, compatible with low-abundance conjugated/complexed TTL species that are more apparent in neuronal tissue.