TOPBP1 Antibody / DNA topoisomerase II-binding protein 1 (FY13094)

Immunofluorescent staining of TOPBP1 using anti-TOPBP1 antibody (green) and anti-Beta Tubulin antibody (red). TOPBP1 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-TOPBP1 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. TOPBP1 displays a punctate nuclear distribution, consistent with its localization to replication and DNA damage–response foci during interphase. The heterogeneous green signal reflects differential concentration within the nucleoplasm, with nucleoli appearing as unstained regions. Beta-tubulin marks the cytoskeleton in red.

Western blot analysis of TOPBP1 using anti-TOPBP1 antibody. Electrophoresis was performed on a 8% SDS-PAGE gel at 80V (Stacking gel) / 120V (Resolving gel) for 2 hours. Lane 1: human Hela whole cell lysates, Lane 2: human HT1080 whole cell lysates, Lane 3: human HEL 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-TOPBP1 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. The expected molecular weight of TOPBP1 is ~170 kDa.

Immunohistochemical staining of TOPBP1 using anti-TOPBP1 antibody. TOPBP1 was detected in a paraffin-embedded section of mouse brain 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-TOPBP1 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.

Immunohistochemical staining of TOPBP1 using anti-TOPBP1 antibody. TOPBP1 was detected in a paraffin-embedded section of rat brain 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-TOPBP1 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 (IP) TOPBP1 in Hela whole cell lysate. Western blot analysis of TOPBP1 using anti-TOPBP1 antibody; Lane 1: Hela whole cell lysates (30ug); Lane 2: Rabbit control IgG instead of anti-TOPBP1 antibody in Hela whole cell lysate; Lane 3: anti-TOPBP1 antibody (2ug) + Hela whole cell lysate (500ug). After electrophoresis, proteins were transferred to a membrane. Then the membrane was incubated with rabbit anti-TOPBP1 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 TOPBP1 at approximately 170 kDa. The expected molecular weight of TOPBP1 is at 170 kDa.