Anti-MBD3 antibody [EPR9913] - ChIP Grade

7 product images

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  ChIP - Anti-MBD3 antibody [EPR9913] - ChIP Grade (ab157464)

  Chromatin was prepared from HeLa cells according to the Abcam Dual X-ChIP protocol*. Cells were fixed with EGS for 30 minutes, then formaldehyde for 10 minutes.
  The ChIP was performed with 25 µg of chromatin, 5 µg of ab157464 (red), and 20 µl of Protein A/G sepharose beads. 5 µg of rabbit normal IgG was added to the beads control (gray). The immunoprecipitated DNA was quantified by real time PCR (Sybr green approach).
  Primers and probes are located in the first kb of the transcribed region.
  *http://www.abcam.com/resources?keywords=X%20ChIP%20protocol

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  ChIP - Anti-MBD3 antibody [EPR9913] - ChIP Grade (ab157464)

  Chromatin was prepared from NIH/3T3 cells according to the Abcam Dual X-ChIP protocol*. Cells were fixed with EGS for 30 minutes, then formaldehyde for 10 minutes.
  The ChIP was performed with 25 µg of chromatin, 5 µg of ab157464 (red), and 20 µl of Protein A/G sepharose beads. 5 µg of rabbit normal IgG was added to the beads control (gray). The immunoprecipitated DNA was quantified by real time PCR (Sybr green approach).
  Primers and probes are located in the first kb of the transcribed region.
  *http://www.abcam.com/resources?keywords=X%20ChIP%20protocol

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  Western blot - Anti-MBD3 antibody [EPR9913] - ChIP Grade (ab157464)

  Lane 1: Wild-type HAP1 cell lysate (20 μg)
  Lane 2: ProteinX knockout HAP1 cell lysate (20 μg)
  Lane 3: Wild-type HAP1 cell lysate (20 μg)
  Lane 4: ProteinX knockout HAP1 cell lysate (20 μg)
  Lanes 1 - 4: Merged signal (red and green). Green - ab157464 observed at 33 kDa. Red - loading control, Anti-GAPDH antibody [6C5] - Loading Control ab8245, observed at 37 kDa.
  
  ab157464 was shown to recognize MBD3 when MBD3 knockout samples were used, along with additional cross-reactive bands. Wild-type and MBD3 knockout samples were subjected to SDS-PAGE. ab157464 and Anti-GAPDH antibody [6C5] - Loading Control ab8245 (loading control to GAPDH) were diluted 1/1000 and 1/10000 respectively and incubated overnight at 4°C. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preadsorbed (Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preadsorbed (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-MBD3 antibody [EPR9913] - ChIP Grade (ab157464)

  Predicted band size: 33 kDa

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  Immunocytochemistry/ Immunofluorescence - Anti-MBD3 antibody [EPR9913] - ChIP Grade (ab157464)

  Immunocytochemistry/Immunofluorescence analysis of HeLa (human cervix adenocarcinoma) labelling MBD3 with purified ab157464 at 1/1000. Cells were fixed with 4% PFA and permeabilized with 0.1% Triton X-100. An Alexa Fluor^® 488-conjugated goat anti-rabbit IgG (1/1000) was used as the secondary antibody (Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) ab150077). Nuclei counterstained with DAPI (blue).
  

  Control: PBS only

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  Western blot - Anti-MBD3 antibody [EPR9913] - ChIP Grade (ab157464)

  All lanes: Western blot - Anti-MBD3 antibody [EPR9913] - ChIP Grade (ab157464) at 1/1000 dilution

  Lane 1: HeLa cell lysate at 10 µg

  Lane 2: 293T cell lysate at 10 µg

  Lane 3: Fetal brain lysate at 10 µg

  Lane 4: Y79 cell lysate at 10 µg

  Secondary

  All lanes: HRP labelled goat anti-rabbit at 1/2000 dilution

  Predicted band size: 33 kDa

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  Immunocytochemistry/ Immunofluorescence - Anti-MBD3 antibody [EPR9913] - ChIP Grade (ab157464)

  Immunofluorescent analysis of HeLa cells labeling MBD3 with ab157464 at 1/100 dilution.

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  Flow Cytometry (Intracellular) - Anti-MBD3 antibody [EPR9913] - ChIP Grade (ab157464)

  Intracellular flow cytometric analysis of permeabilized 293T cells labeling MBD3 with ab157464 at 1/10 dilution (red) compared to a rabbit IgG negative control (green).