MED28 Antibody / Mediator complex subunit 28 (FY12612)

Flow Cytometry analysis of THP-1 cells using anti-MED28 antibody. Overlay histogram showing THP-1 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-MED28 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 (Red line) was also used as a control.

Western blot analysis of MED28 using anti-MED28 antibody. Lane 1: human HEL whole cell lysates, Lane 2: human THP-1 whole cell lysates, Lane 3: human K562 whole cell lysates, Lane 4: 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-MED28 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. Western blot probed with anti-MED28 shows a major band at ~24 kDa, slightly higher than the predicted ~20 kDa, consistent with the known anomalous migration of this small, acidic mediator complex subunit.

Immunoprecipitating MED28 in HepG2 whole cell lysate. Western blot analysis of MED28 using anti-MED28 antibody; Lane 1: HepG2 whole cell lysates (30ug); Lane 2: Rabbit control IgG instead of anti-MED28 antibody in HepG2 whole cell lysate; Lane 3: anti-MED28 antibody (2ug) + HepG2 whole cell lysate (500ug). After electrophoresis, proteins were transferred to a membrane. Then the membrane was incubated with rabbit anti-MED28 antibody at a dilution of 0.5 ug/ml and probed with a goat anti-rabbit IgG-HRP secondary antibody. The signal is developed using ECL Plus Western Blotting Substrate. Western blot probed with anti-MED28 shows a major band at ~24 kDa, slightly higher than the predicted ~20 kDa, consistent with the known anomalous migration of this small, acidic mediator complex subunit.