重组Anti-Dnmt3a抗体[EPR18455]

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-Dnmt3a antibody [EPR18455] (AB188470)

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized HeLa (Human epithelial cells from cervix adenocarcinoma) cells labeling Dnmt3a with ab188470 at 1/1000 dilution, followed by Goat anti-rabbit IgG (Alexa Fluor® 488) (ab150077) secondary antibody at 1/1000 dilution (green). Confocal image showing nuclear and weakly cytoplasmic staining on HeLa cell line. The nuclear counter stain is DAPI (blue). Tubulin is detected with ab7291 (anti-Tubulin mouse mAb) at 1/1000 dilution and ab150120 (AlexaFluor®594 Goat anti-Mouse secondary) at 1/1000 dilution (red).

The negative controls are as follows :
-ve control 1 : ab188470 at 1/1000 dilution followed by ab150120 (AlexaFluor®594 Goat anti-Mouse secondary) at 1/1000 dilution.
-ve control 2 : ab7291 (anti-Tubulin mouse mAb) at 1/1000 dilution followed by ab150077 (Alexa Fluor®488 Goat Anti-Rabbit IgG H&L) at 1/1000 dilution.

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Dnmt3a antibody [EPR18455] (AB188470)

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human placenta tissue sections labeling Dnmt3a with purified ab188470 at 1/2000 (0.409 μg/ml). Antigen retrieval was heat mediated using ab93684 (Tris/EDTA buffer, pH 9.0). Goat Anti-Rabbit IgG H&L (HRP) was used as the secondary antibody. Hematoxylin was used as a counterstain.

• Flow Cyt (Intra)

Lab

Flow Cytometry (Intracellular) - Anti-Dnmt3a antibody [EPR18455] (AB188470)

Intracellular Flow Cytometry analysis of HeLa (human cervix adenocarcinoma) cells labeling Dnmt3a with purified ab188470 at 1/80 dilution (10ug/ml) (red). Cells were fixed with 4% paraformaldehyde and permeabilised with 90% methanol. A Goat anti rabbit IgG (Alexa Fluorr^® 488) (1/2000 dilution) was used as the secondary antibody. Rabbit monoclonal IgG (Black) was used as the isotype control, cells without incubation with primary antibody and secondary antibody (Blue) were used as the unlabeled control.

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Dnmt3a antibody [EPR18455] (AB188470)

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of rat spleen tissue sections labeling Dnmt3 with purified ab188470 at 1/2000 (0.409 μg/ml). Antigen retrieval was heat mediated using ab93684 (Tris/EDTA buffer, pH 9.0). Goat Anti-Rabbit IgG H&L (HRP) was used as the secondary antibody. Hematoxylin was used as a counterstain.

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Dnmt3a antibody [EPR18455] (AB188470)

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of mouse testis tissue sections labeling Dnmt3a with purified ab188470 at 1/2000 (0.409 μg/ml). Antigen retrieval was heat mediated using ab93684 (Tris/EDTA buffer, pH 9.0). Goat Anti-Rabbit IgG H&L (HRP) was used as the secondary antibody. Hematoxylin was used as a counterstain.

• WB

Supplier Data

Western blot - Anti-Dnmt3a antibody [EPR18455] (AB188470)

Blocking/Dilution buffer : 5% NFDM/TBST.

The observed MW is consistent with what has been described in the literature (J Biol Chem. 2002. 277, 38746-38754. PMID : 12138111).

All lanes:

Western blot - Anti-Dnmt3a antibody [EPR18455] (ab188470) at 1/10000 dilution

All lanes:

HeLa (Human epithelial cells from cervix adenocarcinoma) cell lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/50000 dilution

Predicted band size: 102 kDa

Observed band size: 130 kDa

false

Exposure time: 5s

• WB

Lab

Western blot - Anti-Dnmt3a antibody [EPR18455] (AB188470)

Lanes 1 - 4 : Merged signal (red and green). Green - ab188470 observed at 125 kDa. Red - loading control, ab8245, observed at 37 kDa.

ab188470 was shown to recognize Dnmt3a when Dnmt3a knockout samples were used, along with additional cross-reactive bands. Wild-type and Dnmt3a knockout samples were subjected to SDS-PAGE. ab188470 and ab8245 (loading control to GAPDH) were diluted to 1/5000 and 1/10000 respectively and incubated overnight at 4°C. 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 1/10000 dilution for 1 hour at room temperature before imaging.

All lanes:

Western blot - Anti-Dnmt3a antibody [EPR18455] (ab188470) at 1/5000 dilution

Lane 1:

Wild-type HAP1 cell lysate at 20 µg

Lane 2:

Dnmt3a knockout HAP1 cell lysate at 20 µg

Lane 3:

HeLa cell lysate at 20 µg

Lane 4:

Mouse brain tissue lysate at 20 µg

Predicted band size: 102 kDa

false

• WB

Lab

Western blot - Anti-Dnmt3a antibody [EPR18455] (AB188470)

Lanes 1-4 : Merged signal (red and green). Green - ab188470 observed at 125 kDa. Red - loading control ab8245 observed at 37 kDa.

ab188470 Anti-Dnmt3a antibody [EPR18455] 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. ab188470 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) were incubated overnight at 4°C at 1 in 2000 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] (ab188470) at 1/2000 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='/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='/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

false

• WB

Lab

Western blot - Anti-Dnmt3a antibody [EPR18455] (AB188470)

Lanes 1 - 4 : Merged signal (red and green). Green - ab188470 observed at 125 kDa. Red - loading control, ab8245, observed at 37 kDa.

This blot was produced using a 4-12% Bis-tris gel under the MOPS buffer system. The gel was run at 200V for 50 minutes before being transferred onto a Nitrocellulose membrane at 30V for 70 minutes. The membrane was then blocked for an hour using Licor blocking buffer before being incubated with ab188470 and ab8245 (loading control) overnight at 4°C. Antibody binding was detected using Goat anti-Rabbit IgG H&L (IRDye® 800CW) preabsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye® 680RD) preabsorbed (ab216776) at a 1 : 10000 dilution for 1hr at room temperature and then imaged.

All lanes:

Western blot - Anti-Dnmt3a antibody [EPR18455] (ab188470) at 1/5000 dilution

Lane 1:

HeLa whole cell lysate at 20 µg

Lane 2:

Mouse skin tissue lysate at 20 µg

Lane 3:

Rat skin tissue lysate at 20 µg

Lane 4:

Mouse brain tissue lysate at 20 µg

Secondary

All lanes:

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

Predicted band size: 102 kDa

Observed band size: 125 kDa

false

• WB

Supplier Data

Western blot - Anti-Dnmt3a antibody [EPR18455] (AB188470)

Blocking/Dilution buffer : 5% NFDM/TBST.

The observed MW is consistent with what has been described in the literature (J Biol Chem. 2002. 277, 38746-38754. PMID : 12138111).

All lanes:

Western blot - Anti-Dnmt3a antibody [EPR18455] (ab188470) at 1/10000 dilution

All lanes:

HEK-293 (Human epithelial cells from embryonic kidney) cell lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/50000 dilution

Predicted band size: 102 kDa,83 kDa

Observed band size: 130 kDa,74 kDa

false

Exposure time: 15s

• ChIC/CUT&RUN-seq

Supplier Data

ChIC/CUT&RUN sequencing - Anti-Dnmt3a antibody [EPR18455] (AB188470)

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 ab188470, 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

Supplier Data

Western blot - Anti-Dnmt3a antibody [EPR18455] (AB188470)

Blocking/Dilution buffer : 5% NFDM/TBST.

All lanes:

Western blot - Anti-Dnmt3a antibody [EPR18455] (ab188470) at 1/2000 dilution

Lane 1:

Rat brain lysate at 10 µg

Lane 2:

Rat heart lysate at 10 µg

Lane 3:

C6 (Rat glial tumor cells) cell lysate at 10 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/50000 dilution

Predicted band size: 102 kDa

Observed band size: 130 kDa

false

Exposure time: 30s

• WB

CiteAb

Western blot - Anti-Dnmt3a antibody [EPR18455] (AB188470)

Dnmt3a western blot using anti-Dnmt3a antibody [EPR18455] ab188470. Publication image and figure legend from Lei, L., Wu, X., et al., 2020, Front Neurosci, PubMed 33192258.

ab188470 was used in this publication in western blot. This may not be the same as the application(s) guaranteed by Abcam. For a full list of applications guaranteed by Abcam for ab188470 please see the product overview.

Effect of PS on DNMT and demethylase in adolescence. (A) DNMT3a protein expression was markedly upregulated (*p < 0.05, n = 6) while DNMT1 level was marginally increased in PS females. No significant difference in Tet-2 expression was observed between PS and control females. (B) Tet-2 level was decreased in PS males (*p < 0.05, n = 6), whereas DNMT3a and DNMT1 levels were similar in PS and control males. *p < 0.05, *p < 0.01 vs. corresponding control group. Data represent mean ± SEM.

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