POLD1 Antibody / DNA polymerase delta catalytic subunit (FY12910)

Immunofluorescent staining of POLD1 using anti-POLD1 antibody (green) and anti-Beta Tubulin antibody (red). POLD1 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-POLD1 antibody and mouse anti-Beta Tubulin antibody overnight at 4oC. DyLight 488 Conjugated Goat Anti-Rabbit IgG and Cy3 Conjugated Goat Anti-Mouse IgG were used as secondary antibody at 1:500 dilution and incubated for 30 minutes at 37oC. Visualize using a fluorescence microscope and filter sets appropriate for the label used.

Western blot analysis of POLD1 using anti-POLD1 antibody. Electrophoresis was performed on a 8% SDS-PAGE gel at 80V (Stacking gel) / 120V (Resolving gel) for 2 hours. Lane 1: human 293T whole cell lysates, Lane 2: human K562 whole cell lysates, Lane 3: human U2OS whole cell lysates, Lane 4: human Jurkat whole cell lysates, Lane 5: rat thymus tissue lysates, Lane 6: rat spleen tissue lysates, Lane 7: mouse thymus 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-POLD1 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 an ECL Plus Western Blotting Substrate. A specific band was detected for POLD1 at approximately 124 kDa. The expected molecular weight of POLD1 is ~124 kDa.

Immunohistochemical staining of POLD1 using anti-POLD1 antibody. POLD1 was detected in a paraffin-embedded section of human bladder cancer tissue. Heat mediated antigen retrieval was performed in EDTA buffer (pH 8.0, epitope retrieval solution). The tissue section was blocked with 10% goat serum. The tissue section was then incubated with 2 ug/ml rabbit anti-POLD1 antibody overnight at 4oC. Peroxidase Conjugated Goat Anti-rabbit IgG was used as secondary antibody and incubated for 30 minutes at 37oC. The tissue section was developed using an HRP secondary and DAB substrate.

Immunoprecipitating POLD1 in 293T whole cell lysate. Western blot analysis of POLD1 using anti-POLD1 antibody. Lane 1: 293T whole cell lysates (30ug), Lane 2: Rabbit control IgG instead of anti-POLD1 antibody in 293T whole cell lysate, Lane 3: anti-POLD1 antibody (2ug) + 293T whole cell lysate (500ug). After electrophoresis, proteins were transferred to a membrane. Then the membrane was incubated with rabbit anti-POLD1 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 POLD1 at approximately 124 kDa. The expected molecular weight of POLD1 is at 124 kDa.

Flow Cytometry analysis of 293T cells using anti-POLD1 antibody. Overlay histogram showing 293T 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-POLD1 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.