Carf Antibody / Collaborator of ARF / Cdkn2aip (FY13379)

Flow Cytometry analysis of mouse NIH/3T3 cells using anti-Carf antibody. Overlay histogram showing NIH/3T3 cells stained with (Blue line). To facilitate intracellular staining, cells were fixed with 4% paraformaldehyde and permeabilized with permeabilization buffer. The cells were blocked with 10% normal goat serum. And then incubated with rabbit anti-Carf 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 CDKN2AIP/CARF using anti-Carf antibody. Lane 1: rat brain tissue lysates, Lane 2: rat lung tissue lysates, Lane 3: rat kidney tissue lysates, Lane 4: mouse brain tissue lysates, Lane 5: mouse lung tissue lysates, Lane 6: mouse kidney 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-Carf 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. A strong band was detected at approximately 75 kDa in both rat and mouse brain, whereas lung and kidney samples displayed a slightly faster-migrating band at approximately 70 kDa. This upward mobility shift relative to the predicted 61 kDa is consistent with the known phosphorylation-dependent migration behavior of CARF in different tissues.