Anti-DNMT3A Antibody Picoband®

Western blot analysis of DNMT3A using anti-DNMT3A antibody (A00136-2).
Electrophoresis was performed on a 10% SDS-PAGE gel at 80V (Stacking gel) / 120V (Resolving gel) for 2 hours. The sample well of each lane was loaded with 30 ug of sample under reducing conditions.
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 antigen affinity purified polyclonal antibody (A00136-2) at 0.5 μg/ml overnight at 4°C, 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 is developed using an ECL Plus Western Blotting Substrate (Catalog # AR1196-200) with Tanon 5200 system. A specific band was detected for DNMT3A at approximately 120-130 kDa. The expected band size for DNMT3A is at 102 kDa.

IHC analysis of DNMT3A using anti-DNMT3A antibody (A00136-2).
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 μg/ml rabbit anti-DNMT3A Antibody (A00136-2) overnight at 4°C. Peroxidase Conjugated Goat Anti-rabbit IgG was used as secondary antibody and incubated for 30 minutes at 37°C. The tissue section was developed using HRP Conjugated Rabbit IgG Super Vision Assay Kit (Catalog # SV0002) with DAB as the chromogen.

IF analysis of DNMT3A using anti-DNMT3A antibody (A00136-2) and anti-Beta Tubulin antibody (M01857-3).
DNMT3A was detected in an immunocytochemical section of Hela cells. Enzyme antigen retrieval was performed using IHC enzyme antigen retrieval reagent (AR0022) for 15 mins. The cells were blocked with 10% goat serum. And then incubated with 5 μg/mL rabbit anti-DNMT3A Antibody (A00136-2) and mouse anti-Beta Tubulin antibody (M01857-3) overnight at 4°C. DyLight®488 Conjugated Goat Anti-Rabbit IgG (BA1127) and DyLight®550 Conjugated Goat Anti-Mouse IgG (BA1133) were used as secondary antibody at 1:500 dilution and incubated for 30 minutes at 37°C. 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 (A00136-2).
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 (2μg) + MCF-7 whole cell lysate (500μg).
After electrophoresis, proteins were transferred to a membrane. Then the membrane was incubated with rabbit anti-DNMT3A antigen affinity purified polyclonal antibody (A00136-2) at a dilution of 0.5 μg/mL and probed with a goat anti-rabbit IgG-HRP secondary antibody (Catalog # BA1054). The signal is developed using ECL Plus Western Blotting Substrate (Catalog # AR1197). A specific band was detected for DNMT3A at approximately 120-130 kDa. The expected band size for DNMT3A is at 102 kDa.

Flow Cytometry analysis of 293T cells using anti-293T cells antibody (A00136-2).
Overlay histogram showing 293T cells stained with A00136-2 (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 (A00136-2, 1 μg/1x10⁶ cells) for 30 min at 20°C. DyLight®488 conjugated goat anti-rabbit IgG (BA1127, 5-10 μg/1x10⁶ cells) was used as secondary antibody for 30 minutes at 20°C. Isotype control antibody (Green line) was rabbit IgG (1 μg/1x10⁶) used under the same conditions. Unlabelled sample without incubation with primary antibody and secondary antibody (Red line) was used as a blank control.