POLDIP2 Antibody / Polymerase delta-interacting protein 2 (FY13166)

Immunofluorescent staining of POLDIP2 using anti-POLDIP2 antibody (green). POLDIP2 was detected in an immunocytochemical section of U2OS cells. Enzyme antigen retrieval was performed using IHC enzyme antigen retrieval reagent for 15 mins. The cells were blocked with 10% goat serum. And then incubated with 5 ug/ml rabbit anti-POLDIP2 antibody overnight at 4oC. DyLight 488 Conjugated Goat Anti-Rabbit IgG was used as secondary antibody at 1:500 dilution and incubated for 30 minutes at 37oC. The section was counterstained with DAPI nuclear stain (blue). Visualize using a fluorescence microscope and filter sets appropriate for the label used.

Western blot analysis of POLDIP2 using anti-POLDIP2 antibody. Lane 1: human Hela whole cell lysates, Lane 2: human HepG2 whole cell lysates, Lane 3: human Jurkat whole cell lysates, Lane 4: human K562 whole cell lysates, Lane 5: rat testis tissue lysates, Lane 6: rat kidney tissue lysates, Lane 7: mouse testis tissue lysates, Lane 8: 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-POLDIP2 antibody at 0.25 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. POLDIP2 antibody detects a prominent band at ~37 kDa across the indicated lysates. Although the theoretical mass is ~42 kDa, POLDIP2 contains an N-terminal mitochondrial targeting sequence that is cleaved upon import, producing a mature ~37 kDa species that predominates in endogenous samples.

Flow Cytometry analysis of U251 cells using anti-POLDIP2 antibody. Overlay histogram showing U251 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-POLDIP2 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.

Immunoprecipitating POLDIP2 in HepG2 whole cell lysate. Western blot analysis of POLDIP2 using anti-POLDIP2 antibody; Lane 1: HepG2 whole cell lysates (30ug); Lane 2: Rabbit control IgG instead of anti-POLDIP2 antibody in HepG2 whole cell lysate; Lane 3: anti-POLDIP2 antibody (2ug) + HepG2 whole cell lysate (500ug). After electrophoresis, proteins were transferred to a membrane. Then the membrane was incubated with rabbit anti-POLDIP2 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. A specific band was detected for POLDIP2 at approximately 38 kDa. The expected molecular weight of POLDIP2 is at 42 kDa.