Anti-Dnmt3a antibody [EPR26805-273] - BSA and Azide free

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-Dnmt3a antibody [EPR26805-273] - BSA and Azide free (AB307504)

This data was developed using ab307503, the same antibody clone in a different buffer formulation.

Flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized NCCIT (human pluripotent embryonic carcinoma epithelial cell) cells labelling Dnmt3a with ab307503 at 1/500 dilution (0.1ug) (Red) compared with a Rabbit monoclonal IgG (ab172730) (Black) isotype control and an unlabelled 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.

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-Dnmt3a antibody [EPR26805-273] - BSA and Azide free (AB307504)

This data was developed using ab307503, the same antibody clone in a different buffer formulation. Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized NCCIT (human pluripotent embryonic carcinoma epithelial cell) cells labelling Dnmt3a with ab307503 at 1/100 (5.3 ug/ml) dilution, followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed antibody at 1/1000 2 ug/ml dilution (Green). Confocal image showing nuclear staining in NCCIT cell line. Image was taken with a confocal microscope(Leica-Microsystems, TCS SP8). is observed. ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor® 594) was used to counterstain tubulin at 1/200 2.5ug/ml dilution (Red). The 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 2 ug/ml dilution.

• IP

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Immunoprecipitation - Anti-Dnmt3a antibody [EPR26805-273] - BSA and Azide free (AB307504)

This data was developed using ab307503, the same antibody clone in a different buffer formulation.

Dnmt3a was immunoprecipitated from 0.35 mg HeLa (human cervix adenocarcinoma epithelial cell) whole cell lysate 10 ug with ab307503 at 1/30 dilution (2ug 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 (human cervix adenocarcinoma epithelial cell) whole cell lysate 10 ug
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

All lanes:

Immunoprecipitation - Anti-Dnmt3a antibody [EPR26805-273] (<a href='/en-us/products/primary-antibodies/dnmt3a-antibody-epr26805-273-ab307503'>ab307503</a>) at 1/1000 dilution

Lane 1:

HeLa (human cervix adenocarcinoma epithelial cell) whole cell lysate 10 μg

Lane 2:

HeLa whole cell lysate

Lane 3:

Rabbit monoclonal IgG (<a href='/en-us/products/primary-antibodies/rabbit-igg-monoclonal-epr25a-isotype-control-ab172730'>ab172730</a>) instead of <a href='/en-us/products/primary-antibodies/dnmt3a-antibody-epr26805-273-ab307503'>ab307503</a> in Hela whole cell lysate

Secondary

All lanes:

Immunoprecipitation - VeriBlot for IP Detection Reagent (HRP) (<a href='/en-us/products/reagents/veriblot-for-ip-detection-reagent-hrp-ab131366'>ab131366</a>) at 1/5000 dilution

Observed band size: 125 kDa

false

Exposure time: 180s

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Dnmt3a antibody [EPR26805-273] - BSA and Azide free (AB307504)

This data was developed using ab307503, the same antibody clone in a different buffer formulation. Immunohistochemical analysis of paraffin-embedded Rat testis tissue labeling Dnmt3a with ab307503 at 1/1000 (0.532 ug/ml) followed by a ready to use LeicaDS9800 (Bond Polymer Refine Detection) was used. 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 a ready to use LeicaDS9800 (Bond Polymer Refine Detection) was used. Heat mediated antigen retrieval was performed with Citrate buffer (pH 6.0, Epitope Retrieval Solution 1) for 20 mins

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Dnmt3a antibody [EPR26805-273] - BSA and Azide free (AB307504)

This data was developed using ab307503, the same antibody clone in a different buffer formulation. Immunohistochemical analysis of paraffin-embedded Mouse testis tissue labeling Dnmt3a with ab307503 at 1/1000 (0.532 ug/ml) followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection) was used. 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 a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection) was used. Heat mediated antigen retrieval was performed with Citrate buffer (pH 6.0, Epitope Retrieval Solution 1) for 20 mins

• IP

Supplier Data

Immunoprecipitation - Anti-Dnmt3a antibody [EPR26805-273] - BSA and Azide free (AB307504)

This data was developed using ab307503, the same antibody clone in a different buffer formulation. Dnmt3a was immunoprecipitated from 0.35 mg Mouse brain tissue lysate 10 ug with ab307503 at 1/30 dilution (2ug 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 ug 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

All lanes:

Immunoprecipitation - Anti-Dnmt3a antibody [EPR26805-273] (<a href='/en-us/products/primary-antibodies/dnmt3a-antibody-epr26805-273-ab307503'>ab307503</a>) at 1/1000 dilution

Lane 1:

Mouse brain tissue lysate 10 μg

Lane 2:

Mouse brain tissue lysate

Lane 3:

Rabbit monoclonal IgG (<a href='/en-us/products/primary-antibodies/rabbit-igg-monoclonal-epr25a-isotype-control-ab172730'>ab172730</a>) instead of <a href='/en-us/products/primary-antibodies/dnmt3a-antibody-epr26805-273-ab307503'>ab307503</a> in mouse brain tissue lysate

Secondary

All lanes:

Immunoprecipitation - VeriBlot for IP Detection Reagent (HRP) (<a href='/en-us/products/reagents/veriblot-for-ip-detection-reagent-hrp-ab131366'>ab131366</a>) at 1/5000 dilution

Observed band size: 125 kDa

false

Exposure time: 93s

• WB

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Western blot - Anti-Dnmt3a antibody [EPR26805-273] - BSA and Azide free (AB307504)

This data was developed using 307503, the same antibody clone in a different buffer formulation.

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.

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 (ab261793). 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.

All lanes:

Western blot - Anti-Dnmt3a antibody [EPR26805-273] (<a href='/en-us/products/primary-antibodies/dnmt3a-antibody-epr26805-273-ab307503'>ab307503</a>) at 1/1000 dilution

Lane 1:

Wild-type Hela whole cell lysate 20 μg

Lane 2:

Western blot - Human DNMT3A knockout HeLa cell line (<a href='/en-us/products/cell-lines/human-dnmt3a-knockout-hela-cell-line-ab261793'>ab261793</a>)

Lane 2:

Dnmt3a knockout Hela whole cell lysate 20 μg

Lane 3:

Mouse brain tissue lysate 20 μg

Secondary

Lanes 1 - 3:

Western blot - Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-irdye-800cw-preadsorbed-ab216773'>ab216773</a>) at 1/10000 dilution

Lanes 1 - 3:

Western blot - Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed (<a href='/en-us/products/secondary-antibodies/goat-mouse-igg-h-l-irdye-680rd-preadsorbed-ab216776'>ab216776</a>) at 1/20000 dilution

Observed band size: 125 kDa

false

• WB

Supplier Data

Western blot - Anti-Dnmt3a antibody [EPR26805-273] - BSA and Azide free (AB307504)

This data was developed using 307503, the same antibody clone in a different buffer formulation. Blocking and diluting buffer and concentration : 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. 103 seconds Exposure time :

All lanes:

Western blot - Anti-Dnmt3a antibody [EPR26805-273] (<a href='/en-us/products/primary-antibodies/dnmt3a-antibody-epr26805-273-ab307503'>ab307503</a>) at 1/1000 dilution

Lane 1:

HEK-293 (human embryonic kidney epithelial cell) whole cell lysate 20 μg

Lane 2:

C6 (rat glial tumor glial cell) whole cell lysate 20 μg

Lane 3:

Mouse brain tissue lysate 20 μg

Lane 4:

NCCIT (human pluripotent embryonic carcinoma epithelial cell) whole cell lysate 20 μg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/100000 dilution

Observed band size: 85 kDa,125 kDa

false

Exposure time: 103s

• ChIC/CUT&RUN-seq

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ChIC/CUT&RUN sequencing - Anti-Dnmt3a antibody [EPR26805-273] - BSA and Azide free (AB307504)

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab307503)

CUT&RUN profiling with Dnmt3a antibody reveals the expected genomic enrichment pattern in cells. Representative genome browser tracks show CUT&RUN data generated using the CUTANA™ CUT&RUN Kit (EpiCypher 14-1048) with Dnmt3a antibody (Abcam ab307503, 0.5 µg). 500,000 HeLa cells were used per reaction. IgG, H3K4me3, and H3K27me3 antibodies were included as controls to assess non-specific background, active promoters, and repressed chromatin, respectively. Libraries were prepared using the CUTANA™ CUT&RUN Library Prep Kit (EpiCypher 14-1001). Sequencing was performed with paired-end 50 bp reads, and data were processed on CUTANA™ Cloud (cloud.epicypher.com) by alignment to the hg38 genome. Images were generated using Integrative Genomics Viewer (IGV, Broad Institute).