Anti-Dnmt3a antibody [EPR18455-94]

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Dnmt3a antibody [EPR18455-94] (AB227823)

Immunohistochemical analysis of paraffin-embedded human placenta tissue labeling Dnmt3a with ab227823 at 1/200 dilution, followed by Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Nuclear staining on the human placenta (PMID : 22413869) is observed. Counter stained with hematoxylin.

Secondary antibody only control : Used PBS instead of primary antibody, secondary antibody is Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution 2) for 20 minutes.

The section was incubated with ab227823 for 30 mins at room temperature.

The immunostaining was performed on a Leica Biosystems BOND^® RX instrument.

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Dnmt3a antibody [EPR18455-94] (AB227823)

Immunohistochemical analysis of paraffin-embedded human breast carcinoma tissue labeling Dnmt3a with ab227823 at 1/200 dilution, followed by Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Nuclear staining on the human breast carcinoma (PMID : 24464625) is observed. Counter stained with hematoxylin.

Secondary antibody only control : Used PBS instead of primary antibody, secondary antibody is Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution 2) for 20 minutes.

The section was incubated with ab227823 for 30 mins at room temperature.

The immunostaining was performed on a Leica Biosystems BOND^® RX instrument.

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Dnmt3a antibody [EPR18455-94] (AB227823)

Immunohistochemical analysis of paraffin-embedded mouse colon tissue labeling Dnmt3a with ab227823 at 1/200 dilution, followed by Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Nuclear staining on the mouse colon (PMID : 24837369) is observed. Counter stained with hematoxylin.

Secondary antibody only control : Used PBS instead of primary antibody, secondary antibody is Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution 2) for 20 minutes.

The section was incubated with ab227823 for 30 mins at room temperature.

The immunostaining was performed on a Leica Biosystems BOND^® RX instrument.

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Dnmt3a antibody [EPR18455-94] (AB227823)

Immunohistochemical analysis of paraffin-embedded rat colon tissue labeling Dnmt3a with ab227823 at 1/200 dilution, followed by Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Nuclear staining on the rat colon (PMID : 24837369) is observed. Counter stained with hematoxylin.

Secondary antibody only control : Used PBS instead of primary antibody, secondary antibody is Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution 2) for 20 minutes.

The section was incubated with ab227823 for 30 mins at room temperature.

The immunostaining was performed on a Leica Biosystems BOND^® RX instrument.

• WB

Unknown

Western blot - Anti-Dnmt3a antibody [EPR18455-94] (AB227823)

Blocking and dilution buffer : 5% NFDM/TBST.

ab227823 was shown to specifically react with wild-type HAP1 cells as signal was lost in Dnmt3a knockout cells. Wild-type and Dnmt3a knockout samples were subjected to SDS-PAGE. ab227823 and ab181602 (Rabbit anti-GAPDH loading control) were incubated 1 hour at room temperature at 1/1000 dilution and 1/200,000 dilution respectively. Blots were developed with Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (ab97051) secondary antibody at 1/100,000 dilution for 1 hour at room temperature before imaging. The blot was developed on a BIO-RAD® ChemiDoc™ MP instrument using the ECL technique.

The observed bands ranging from 75~125kDa represent different Dnmt3a isoforms.

All lanes:

Western blot - Anti-Dnmt3a antibody [EPR18455-94] (ab227823) at 1/1000 dilution

Lane 1:

Wild-type HAP1 whole cell lysate at 20 µg

Lane 2:

Dnmt3a knockout HAP1 whole cell lysate at 20 µg

Lane 3:

HEK-293T (human epithelial cell line from embryonic kidney transformed with large T antigen) whole cell lysate at 20 µg

Lane 4:

HeLa (human epithelial cell line from cervix adenocarcinoma) whole cell lysate at 20 µg

Lane 5:

NIH/3T3 (mouse embryo fibroblast cell line) whole cell lysate at 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

Predicted band size: 102 kDa

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• WB

Lab

Western blot - Anti-Dnmt3a antibody [EPR18455-94] (AB227823)

Lanes 1-4 : Merged signal (red and green). Green - ab227823 observed at 125 kDa. Red - loading control ab8245 observed at 37 kDa.

ab227823 Anti-Dnmt3a antibody [EPR18455-94] was shown to specifically react with Dnmt3a in wild-type HeLa cells. Loss of signal was observed when knockout cell line ab261793 (knockout cell lysate ab257128) was used. Wild-type and Dnmt3a knockout samples were subjected to SDS-PAGE. ab227823 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) were incubated overnight at 4°C at 1 in 1000 dilution and 1 in 20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preadsorbed (ab216776) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.

All lanes:

Western blot - Anti-Dnmt3a antibody [EPR18455-94] (ab227823) at 1/1000 dilution

Lane 1:

Wild-type HeLa cell lysate at 20 µg

Lane 2:

Dnmt3a knockout HeLa cell lysate at 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 3:

HEK-293 cell lysate at 20 µg

Lane 4:

Daudi cell lysate at 20 µg

Secondary

All lanes:

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 1/20000 dilution

Predicted band size: 102 kDa

Observed band size: 125 kDa

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• ChIC/CUT&RUN-seq

Supplier Data

ChIC/CUT&RUN sequencing - Anti-Dnmt3a antibody [EPR18455-94] (AB227823)

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 ab227823, 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).

• WB

Unknown

Western blot - Anti-Dnmt3a antibody [EPR18455-94] (AB227823)

Blocking and dilution buffer : 5% NFDM/TBST.

The observed bands ranging from 75~125kDa represent different Dnmt3a isoforms.

All lanes:

Western blot - Anti-Dnmt3a antibody [EPR18455-94] (ab227823) at 1/1000 dilution

All lanes:

Rat brain lysate at 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

Predicted band size: 102 kDa

Observed band size: 125 kDa

false

Exposure time: 26s