Anti-Dnmt3a antibody [EPR26805-273]

8 product images

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  Western blot - 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.

  
  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 (Human DNMT3A knockout HeLa 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 (Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed ab216773) at 1/10000 dilution and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed (Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed ab216776) at 1/20000 dilution.

  All lanes: Western blot - Anti-Dnmt3a antibody [EPR26805-273] (ab307503) at 1/1000 dilution

  Lane 1: Wild-type Hela whole cell lysate 20 μg

  Lane 2: Western blot - Human DNMT3A knockout HeLa cell line (Human DNMT3A knockout HeLa cell line ab261793)

  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 (Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed ab216773) at 1/10000 dilution

  Lanes 1 - 3: Western blot - Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed (Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed ab216776) at 1/20000 dilution

  Performed under reducing conditions.

  Observed band size: 125 kDa

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

  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)(VeriBlot for IP Detection Reagent (HRP) ab131366) was used at 1/5000 dilution. Lane 1: Mouse brain tissue lysate 10 ug
  Lane 2: ABAB307503 IP in Mouse brain tissue lysate
  Lane 3:RABbit monoclonal IgG (Rabbit IgG, monoclonal [EPR25A] - Isotype Control 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] (ab307503) at 1/1000 dilution

  Lane 1: Mouse brain tissue lysate 10 μg

  Lane 2: Mouse brain tissue lysate

  Lane 3: Immunoprecipitation - Rabbit IgG, monoclonal [EPR25A] - Isotype Control (Rabbit IgG, monoclonal [EPR25A] - Isotype Control ab172730)

  Secondary

  All lanes: Immunoprecipitation - VeriBlot for IP Detection Reagent (HRP) (VeriBlot for IP Detection Reagent (HRP) ab131366) at 1/5000 dilution

  Observed band size: 125 kDa

  Exposure time: 93s

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

  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)(VeriBlot for IP Detection Reagent (HRP) ab131366) was used at 1/5000 dilution. Lane 1: HeLa (human cervix adenocarcinoma epithelial cell) whole cell lysate 10 ug
  Lane 2: ABAB307503 IP in HeLa whole cell lysate
  Lane 3:RABbit monoclonal IgG (Rabbit IgG, monoclonal [EPR25A] - Isotype Control 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] (ab307503) 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: Immunoprecipitation - Rabbit IgG, monoclonal [EPR25A] - Isotype Control (Rabbit IgG, monoclonal [EPR25A] - Isotype Control ab172730)

  Secondary

  All lanes: Immunoprecipitation - VeriBlot for IP Detection Reagent (HRP) (VeriBlot for IP Detection Reagent (HRP) ab131366) at 1/5000 dilution

  Observed band size: 125 kDa

  Exposure time: 180s

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  Western blot - Anti-Dnmt3a antibody [EPR26805-273] (ab307503)

  Blocking and diluting buffer and concentration: 5% NFDM/TBSTThe 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

  All lanes: Western blot - Anti-Dnmt3a antibody [EPR26805-273] (ab307503) 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) (Goat Anti-Rabbit IgG H&L (HRP) ab97051) at 1/100000 dilution

  Observed band size: 85 kDa, 125 kDa

  Exposure time: 103s

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  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Dnmt3a antibody [EPR26805-273] (ab307503)

  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

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  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Dnmt3a antibody [EPR26805-273] (ab307503)

  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

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  Immunocytochemistry/ Immunofluorescence - Anti-Dnmt3a antibody [EPR26805-273] (ab307503)

  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 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed 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. Alexa Fluor® 594 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker 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 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed ab150081 Goat Anti-RABbit IgG H&L (Alexa Fluor? 488) preadsorbed at 1/1000 2 ug/ml dilution.

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  Flow Cytometry (Intracellular) - Anti-Dnmt3a antibody [EPR26805-273] (ab307503)

  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) (Red) compared with a RABbit monoclonal IgG (Rabbit IgG, monoclonal [EPR25A] - Isotype Control 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, Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed ab150081) at 1/2000 dilution was used as the secondary antibody.