DNMT3A Antibody / DNA (cytosine-5)-methyltransferase 3A (FY13240)

Immunohistochemical staining of DNMT3A using anti-DNMT3A antibody. DNMT3A was detected in a paraffin-embedded section of human colon 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-DNMT3A 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.

Western blot analysis of DNMT3A using anti-DNMT3A antibody. Electrophoresis was performed on a 10% SDS-PAGE gel at 80V (Stacking gel) / 120V (Resolving gel) for 2 hours. Lane 1: human 293T whole cell lysates, Lane 2: human MCF-7 whole cell lysates, Lane 3: rat testis tissue lysates, Lane 4: mouse testis tissue lysates, Lane 5: mouse NIH/3T3 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-DNMT3A 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. Western blot detection of DNMT3A shows a predominant band at ~120-130 kDa across cell lines, consistent with the DNMT3A1 isoform. The shorter DNMT3A2 isoform (~102 kDa) is not prominent in these samples. Post-translational modifications may further increase apparent molecular weight.

Immunofluorescent staining of DNMT3A using anti-DNMT3A antibody (green) and anti-Beta Tubulin antibody (red). DNMT3A was detected in an immunocytochemical section of Hela 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-DNMT3A antibody and mouse anti-Beta Tubulin antibody overnight at 4oC. DyLight 488 Conjugated Goat Anti-Rabbit IgG and DyLight 550 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.

Immunoprecipitating DNMT3A in MCF-7 whole cell lysate. Western blot analysis of DNMT3A using anti-DNMT3A antibody. Lane 1: MCF-7 whole cell lysates (30ug), Lane 2: Rabbit control IgG instead of anti-DNMT3A antibody in MCF-7 whole cell lysate, Lane 3: anti-DNMT3A antibody (2ug) + MCF-7 whole cell lysate (500ug). After electrophoresis, proteins were transferred to a membrane. Then the membrane was incubated with rabbit anti-DNMT3A 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 detection of DNMT3A shows a predominant band at ~120-130 kDa across cell lines, consistent with the DNMT3A1 isoform. The shorter DNMT3A2 isoform (~102 kDa) is not prominent in this sample. Post-translational modifications may further increase apparent molecular weight.

Flow Cytometry analysis of 293T cells using anti-293T cells 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-293T cells 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.