ARID3A Antibody / AT-rich interactive domain-containing protein 3A (FY12340)

Flow Cytometry analysis of Caco-2 cells using anti-ARID3A antibody. Overlay histogram showing Caco-2 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-ARID3A 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 ARID3A using anti-ARID3A antibody. Lane 1: human K562 whole cell lysates, Lane 2: human HepG2 whole cell 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-ARID3A 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 molecular weight of ARID3A is ~63 kDa, can be observed at 75-80 kDa. Peer-reviewed studies show ARID3A is extensively phosphorylated, SUMOylated, and palmitoylated. These modifications-particularly clustered phosphorylation and SUMO conjugation-slow its migration on SDS-PAGE, explaining why it appears at ~75–80 kDa instead of the predicted ~63 kDa.

Immunoprecipitation of ARID3A in HepG2 whole cell lysate. Western blot analysis of ARID3A using anti-ARID3A antibody. Lane 1: HepG2 whole cell lysates (30ug), Lane 2: Rabbit control IgG instead of anti-ARID3A antibody in HepG2 whole cell lysate, Lane 3: anti-ARID3A antibody (2ug) + HepG2 whole cell lysate (500ug). After electrophoresis, proteins were transferred to a membrane. Then the membrane was incubated with rabbit anti-ARID3A antibody at a dilution of 0.5 ug/ml and probed with a goat anti-rabbit IgG-HRP secondary antibody (Light chain). The signal is developed using ECL Plus Western Blotting Substrate. The expected molecular weight of ARID3A is ~63 kDa, can be observed at 75-80 kDa. Peer-reviewed studies show ARID3A is extensively phosphorylated, SUMOylated, and palmitoylated. These modifications-particularly clustered phosphorylation and SUMO conjugation-slow its migration on SDS-PAGE, explaining why it appears at ~75–80 kDa instead of the predicted ~63 kDa.