Recombinant Anti-Dnmt3a antibody [EPR26805-273] (ab307503)

Blocking and diluting buffer and concentration: Intercept® (TBS) Blocking Buffer diluted with an equal volume of 0.1% TBS. The samples were run on a Bis-Tris gel.

Performed under reducing conditions.

False colour image of Western blot: Anti-Dnmt3a antibody ab307503 staining at 1/1000 dilution, shown in green; Mouse anti-GAPDH antibody 6C5 loading control staining at 1/20000 dilution, shown in red.

In Western blot, ab307503 was shown to bind specifically to Dnmt3a. A band was observed at 125 kDa in wild-type Hela cell lysates whereas no signal observed at this size in Dnmt3a knockout cell line (ab227823). To generate this image, wild-type and Dnmt3a knockout Hela cell lysates were analyzed. First, samples were run on a SDS-PAGE gel then transferred onto an immobilon-FL PVDF membrane. Membranes were blocked in in Intercept® (TBS) Blocking Buffer diluted with an equal volume of 0.1% TBS before incubation with primary antibodies overnight at 4°C. Blots were washed four times in TBS-T, incubated with secondary antibodies for 1 h at room temperature, washed again four times then imaged. Secondary antibodies used were Goat anti-Rabbit IgG H&L (IRDye® 800CW) preabsorbed (ab216773) at 1/10000 dilution and Goat anti-Mouse IgG H&L (IRDye® 680RD) preabsorbed (ab216776) at 1/20000 dilution.

Blocking and diluting buffer and concentration: 5% NFDM/TBST.

The expression profile/ molecular weight observed is consistent with what has been described in the literature (PMID: 31956100).

Lysates were freshly made and used for Western blotting immediately to minimize protein degradation.

Exposure time: 103 seconds.

Immunohistochemical analysis of paraffin-embedded mouse testis tissue labeling Dnmt3a with ab307503 at 1/1000 dilution (0.532 µg/ml) followed by ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).
Nuclear staining on mouse testis.
The section was incubated with ab307503 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND® RX instrument. Counterstained with Hematoxylin.

Secondary antibody only control: Secondary antibody is ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Heat mediated antigen retrieval was performed with Citrate buffer (pH 6.0, Epitope Retrieval Solution 1) for 20 mins.

Immunohistochemical analysis of paraffin-embedded rat testis tissue labeling Dnmt3a with ab307503 at 1/1000 dilution (0.532 µg/ml) followed by ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).
Nuclear staining on rat testis.
The section was incubated with ab307503 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND® RX instrument. Counterstained with Hematoxylin.

Secondary antibody only control: Secondary antibody is ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Heat mediated antigen retrieval was performed with Citrate buffer (pH 6.0, Epitope Retrieval Solution 1) for 20 mins.

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized NCCIT (human pluripotent embryonic carcinoma epithelial cell) cells labeling Dnmt3a with ab307503 at 1/100 dilution (5.3 µg/ml), followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed antibody at 1/1000 dilution (2 µg/ml) (Green).
Confocal image showing nuclear staining in NCCIT cell line.
Image was taken with a confocal microscope(Leica-Microsystems, TCS SP8). ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor® 594) was used to counterstain tubulin at 1/200 dilution (2.5 µg/ml) (Red). Nuclear counterstain was DAPI (Blue).

Secondary antibody only control: Secondary antibody is ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1000 dilution (2 µg/ml).

Flow cytometric analysis of 4% paraformaldehyde-fixed 90% methanol-permeabilized NCCIT (human pluripotent embryonic carcinoma epithelial cell) cells labeling Dnmt3a with ab307503 at 1/500 dilution (0.1 µg) (Red) compared with a Rabbit monoclonal IgG (ab172730) (Black) isotype control and an unlabeled control (cells without incubation with primary antibody and secondary antibody) (Blue). A Goat Anti-Rabbit IgG (Alexa Fluor® 488, ab150081) at 1/2000 dilution was used as the secondary antibody.

Dnmt3a was immunoprecipitated from 0.35 mg mouse brain tissue lysate 10 µg with ab307503 at 1/30 dilution (2µg in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab307503 at 1/1000 dilution. VeriBlot for IP secondary antibody(HRP)(ab131366) was used at 1/5000 dilution.

Lane 1: Mouse brain tissue lysate 10 µg

Lane 2: ab307503 IP in mouse brain tissue lysate

Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab307503 in mouse brain tissue lysate

Blocking and dilution buffer and concentration: 5% NFDM/TBST.

Exposure time: 93 seconds.

Dnmt3a was immunoprecipitated from 0.35 mg HeLa (human cervix adenocarcinoma epithelial cell) whole cell lysate 10 µg with ab307503 at 1/30 dilution (2µg in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab307503 at 1/1000 dilution. VeriBlot for IP secondary antibody(HRP)(ab131366) was used at 1/5000 dilution.

Lane 1: HeLa whole cell lysate 10 µg

Lane 2: ab307503 IP in HeLa whole cell lysate

Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab307503 in HeLa whole cell lysate

Blocking and dilution buffer and concentration: 5% NFDM/TBST.

Exposure time: 180 seconds.