MADD Antibody / MAP kinase-activating death domain (FY12091)

Flow Cytometry analysis of Hela cells using anti-MADD antibody. Overlay histogram showing Hela cells stained with (Blue line). The cells were fixed with 4% paraformaldehyde and blocked with 10% normal goat serum. And then incubated with rabbit anti-MADD antibody (1 ug/million cells) for 30 min at 20oC. DyLight 488 conjugated goat anti-rabbit IgG (5-10 ug/million cells) was used as secondary antibody for 30 minutes at 20oC. Isotype control antibody (Green line) was rabbit IgG (1 ug/million cells) used under the same conditions. Unlabelled sample without incubation with primary antibody and secondary antibody (Red line) was used as a blank control.

Western blot analysis of MADD using anti-MADD antibody. Lane 1: human K562 whole cell lysates, Lane 2: human Hela whole cell lysates, Lane 3: human HEL whole cell lysates, Lane 4: human RT4 whole cell lysates, Lane 5: rat brain tissue lysates, Lane 6: 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-MADD 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 enhanced chemiluminescent. The expected band size for MADD is at 183 kDa but can be observed at 220-250 kDa (and as a possible doublet) due to alternative splicing and phosphorylation.