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    products/primary-antibodies/mbd3-antibody-epr18258-ab188401.pdf

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Epigenetics and Nuclear Signaling DNA methylation Methylated DNA Binding
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Validated using a knockout cell lineRecombinantRabMAb

Recombinant Anti-MBD3 antibody [EPR18258] (ab188401)

  • Datasheet
  • SDS
  • Certificate of Compliance
Submit a review Submit a question References (1)

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Western blot - Anti-MBD3 antibody [EPR18258] (ab188401)
  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-MBD3 antibody [EPR18258] (ab188401)
  • Immunocytochemistry/ Immunofluorescence - Anti-MBD3 antibody [EPR18258] (ab188401)
  • Western blot - Anti-MBD3 antibody [EPR18258] (ab188401)
  • Immunocytochemistry/ Immunofluorescence - Anti-MBD3 antibody [EPR18258] (ab188401)
  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-MBD3 antibody [EPR18258] (ab188401)
  • Western blot - Anti-MBD3 antibody [EPR18258] (ab188401)
  • Western blot - Anti-MBD3 antibody [EPR18258] (ab188401)
  • Western blot - Anti-MBD3 antibody [EPR18258] (ab188401)
  • Anti-MBD3 antibody [EPR18258] (ab188401)

Key features and details

  • Produced recombinantly (animal-free) for high batch-to-batch consistency and long term security of supply
  • Rabbit monoclonal [EPR18258] to MBD3
  • Suitable for: WB, ICC/IF, IHC-P
  • Knockout validated
  • Reacts with: Mouse, Rat, Human

Conjugates logo Related conjugates and formulations

Carrier Free

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Recombinant Human MBD3 protein (ab134609)
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Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) (ab150077)
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Anti-BRCC45/BRE antibody [EPR11858] (ab177960)

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Overview

  • Product name

    Anti-MBD3 antibody [EPR18258]
    See all MBD3 primary antibodies
  • Description

    Rabbit monoclonal [EPR18258] to MBD3
  • Host species

    Rabbit
  • Specificity

    This antibody detects a doublet by western blot, which we believe corresponds to isoforms 1 and 2 of MBD3.

    This product is only recommended for human IHC but not for mouse or rat.

  • Tested applications

    Suitable for: WB, ICC/IF, IHC-Pmore details
  • Species reactivity

    Reacts with: Mouse, Rat, Human
  • Immunogen

    Synthetic peptide. This information is proprietary to Abcam and/or its suppliers.

  • Positive control

    • WB: HeLa, 293, F9, WEHI-3, T-47D, HepG2, C6, RAW 264.7, PC-12 and NIH/3T3 whole cell lysates; Human fetal kidney lysate; Mouse spleen lysate. ICC/IF: HeLa and NIH/3T3 cells.
  • General notes

    This product is a recombinant monoclonal antibody, which offers several advantages including:

    • - High batch-to-batch consistency and reproducibility
    • - Improved sensitivity and specificity
    • - Long-term security of supply
    • - Animal-free production
    For more information see here.

    Our RabMAb® technology is a patented hybridoma-based technology for making rabbit monoclonal antibodies. For details on our patents, please refer to RabMAb® patents.

Properties

  • Form

    Liquid
  • Storage instructions

    Shipped at 4°C. Store at +4°C short term (1-2 weeks). Upon delivery aliquot. Store at -20°C long term. Avoid freeze / thaw cycle.
  • Storage buffer

    pH: 7.2
    Preservative: 0.01% Sodium azide
    Constituents: 59% PBS, 0.05% BSA, 40% Glycerol (glycerin, glycerine)
  • Concentration information loading...
  • Purity

    Protein A purified
  • Clonality

    Monoclonal
  • Clone number

    EPR18258
  • Isotype

    IgG
  • Research areas

    • Epigenetics and Nuclear Signaling
    • DNA methylation
    • Methylated DNA Binding

Associated products

  • Alternative Versions

    • Anti-MBD3 antibody [EPR18258] - BSA and Azide free (ab250961)
  • Compatible Secondaries

    • Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) (ab150077)
    • Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (ab216773)
  • Isotype control

    • Rabbit IgG, monoclonal [EPR25A] - Isotype Control (ab172730)
  • Recombinant Protein

    • Recombinant Human MBD3 protein (ab134609)

Applications

The Abpromise guarantee

Our Abpromise guarantee covers the use of ab188401 in the following tested applications.

The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.

Application Abreviews Notes
WB
1/2000. Detects a band of approximately 33, 29 kDa (predicted molecular weight: 33, 29 kDa).
ICC/IF
1/500.
IHC-P
1/200. Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.
Notes
WB
1/2000. Detects a band of approximately 33, 29 kDa (predicted molecular weight: 33, 29 kDa).
ICC/IF
1/500.
IHC-P
1/200. Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

Target

  • Function

    Does not bind DNA by itself. Recruits histone deacetylases and DNA methyltransferases. Acts as transcriptional repressor and plays a role in gene silencing.
  • Sequence similarities

    Contains 1 MBD (methyl-CpG-binding) domain.
  • Cellular localization

    Nucleus. Nuclear, in discrete foci.
  • Target information above from: UniProt accession O95983 The UniProt Consortium
    The Universal Protein Resource (UniProt) in 2010
    Nucleic Acids Res. 38:D142-D148 (2010) .

    Information by UniProt
  • Database links

    • Entrez Gene: 53615 Human
    • Entrez Gene: 17192 Mouse
    • Entrez Gene: 362834 Rat
    • Omim: 603573 Human
    • SwissProt: O95983 Human
    • SwissProt: Q9Z2D8 Mouse
    • Unigene: 178728 Human
    • Unigene: 7142 Mouse
    • Alternative names

      • AI181826 antibody
      • AU019209 antibody
      • MBD 3 antibody
      • Mbd3 antibody
      • MBD3: methyl CpG binding domain protein 3 antibody
      • MBD3_HUMAN antibody
      • Methyl CpG binding domain protein 3 antibody
      • Methyl CpG binding protein MBD3 antibody
      • Methyl-CpG-binding domain protein 3 antibody
      • Methyl-CpG-binding protein MBD3 antibody
      see all

    Images

    • Western blot - Anti-MBD3 antibody [EPR18258] (ab188401)
      Western blot - Anti-MBD3 antibody [EPR18258] (ab188401)

      Lane 1: Wild-type HAP1 cell lysate (20 µg)
      Lane 2: MBD3 knockout HAP1 cell lysate (20 µg)
      Lane 3: HeLa cell lysate (20 µg)
      Lane 4: F9 cell lysate (20 µg)
      Lanes 1 - 4: Merged signal (red and green). Green - ab188401 observed at 33 kDa. Red - loading control, ab8245, observed at 37 kDa.

      ab188401 was shown to specifically react with MBD3 when MBD3 knockout samples were used. Wild-type and MBD3 knockout samples were subjected to SDS-PAGE. ab188401 and ab8245 (loading control to GAPDH) were diluted 1/5000 and 1/10000 and incubated respectively 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.

    • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-MBD3 antibody [EPR18258] (ab188401)
      Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-MBD3 antibody [EPR18258] (ab188401)

      Immunohistochemical analysis of paraffin-embedded Human stomach tissue labeling MBD3 with ab188401 at 1/200 dilution (6.17 μg/ml) followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Nuclear staining on the human stomach. The section was incubated with ab188401 for 15 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND® RX instrument. Counterstained with Hematoxylin. Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution 2) for 20 mins.

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

    • Immunocytochemistry/ Immunofluorescence - Anti-MBD3 antibody [EPR18258] (ab188401)
      Immunocytochemistry/ Immunofluorescence - Anti-MBD3 antibody [EPR18258] (ab188401)

      Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized HeLa (Human epithelial cells from cervix adenocarcinoma) cells labeling MBD3 with ab188401 at 1/500 dilution, followed by Goat anti-rabbit IgG (Alexa Fluor® 488) (ab150077) secondary antibody at 1/1000 dilution (green). Confocal image showing nuclear 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: ab188401 at 1/500 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.

       

    • Western blot - Anti-MBD3 antibody [EPR18258] (ab188401)
      Western blot - Anti-MBD3 antibody [EPR18258] (ab188401)
      All lanes : Anti-MBD3 antibody [EPR18258] (ab188401) at 1/5000 dilution

      Lane 1 : HeLa (Human epithelial cells from cervix adenocarcinoma) whole cell lysate
      Lane 2 : 293 (Human epithelial cells from embryonic kidney) whole cell lysate
      Lane 3 : F9 (Mouse embyro testicular cancer cell line) whole cell lysate
      Lane 4 : WEHI-3 (Mouse leukemia cell line) whole cell lysate
      Lane 5 : T-47D (Human ductal breast epithelial tumor cell line) whole cell lysate
      Lane 6 : HepG2 (Human liver hepatocellular carcinoma) whole cell lysate

      Lysates/proteins at 10 µg per lane.

      Secondary
      All lanes : Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated at 1/1000 dilution

      Predicted band size: 33, 29 kDa
      Observed band size: 29,33 kDa why is the actual band size different from the predicted?


      Exposure time: 1 minute


      Blocking/Dilution buffer: 5% NFDM/TBST.

      The 33 and 29 kDa bands correspond to Isoforms 1 and 2.

    • Immunocytochemistry/ Immunofluorescence - Anti-MBD3 antibody [EPR18258] (ab188401)
      Immunocytochemistry/ Immunofluorescence - Anti-MBD3 antibody [EPR18258] (ab188401)

      Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized NIH/3T3 (Mouse embyro fibroblast cells) cells labeling MBD3 with ab188401 at 1/500 dilution, followed by Goat anti-rabbit IgG (Alexa Fluor® 488) (ab150077) secondary antibody at 1/1000 dilution (green). Confocal image showing nuclear staining on NIH/3T3 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: ab188401 at 1/500 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.

       

    • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-MBD3 antibody [EPR18258] (ab188401)
      Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-MBD3 antibody [EPR18258] (ab188401)

      Immunohistochemical analysis of paraffin-embedded Human pancreas tissue labeling MBD3 with ab188401 at 1/200 dilution (6.17 μg/ml) followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Nuclear staining on the human pancreas (PMID: 27898661). The section was incubated with ab188401 for 15 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND® RX instrument. Counterstained with Hematoxylin. Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution 2) for 20 mins.

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

    • Western blot - Anti-MBD3 antibody [EPR18258] (ab188401)
      Western blot - Anti-MBD3 antibody [EPR18258] (ab188401)
      All lanes : Anti-MBD3 antibody [EPR18258] (ab188401) at 1/2000 dilution

      Lane 1 : C6 (Rat glial tumor cells) whole cell lysate
      Lane 2 : RAW 264.7 (Mouse macrophage cells transformed with Abelson murine leukemia virus) whole cell lysate
      Lane 3 : PC-12 (Rat adrenal gland pheochromocytoma) whole cell lysate
      Lane 4 : NIH/3T3 (Mouse embyro fibroblast cells) whole cell lysate

      Lysates/proteins at 10 µg per lane.

      Secondary
      All lanes : Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated at 1/1000 dilution

      Predicted band size: 33, 29 kDa
      Observed band size: 29,33 kDa why is the actual band size different from the predicted?


      Exposure time: 15 seconds


      Blocking/Dilution buffer: 5% NFDM/TBST.

      The 33 and 29 kDa bands correspond to Isoforms 1 and 2.

    • Western blot - Anti-MBD3 antibody [EPR18258] (ab188401)
      Western blot - Anti-MBD3 antibody [EPR18258] (ab188401)
      Anti-MBD3 antibody [EPR18258] (ab188401) at 1/2000 dilution + Human fetal kidney tissue lysate at 10 µg

      Secondary
      Anti-Rabbit IgG (HRP), specific to the non-reduced form of IgG at 1/1000 dilution

      Predicted band size: 33, 29 kDa
      Observed band size: 29,33 kDa why is the actual band size different from the predicted?


      Exposure time: 3 minutes


      Blocking/Dilution buffer: 5% NFDM/TBST.

    • Western blot - Anti-MBD3 antibody [EPR18258] (ab188401)
      Western blot - Anti-MBD3 antibody [EPR18258] (ab188401)
      Anti-MBD3 antibody [EPR18258] (ab188401) at 1/2000 dilution + Mouse spleen tissue lysate at 10 µg

      Secondary
      Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated at 1/1000 dilution

      Predicted band size: 33, 29 kDa
      Observed band size: 29,33 kDa why is the actual band size different from the predicted?


      Exposure time: 3 minutes


      Blocking/Dilution buffer: 5% NFDM/TBST.

      The 33 and 29 kDa bands correspond to Isoforms 1 and 2.

    • Anti-MBD3 antibody [EPR18258] (ab188401)
      Anti-MBD3 antibody [EPR18258] (ab188401)

    Protocols

    • Western blot protocols
    • Immunocytochemistry & immunofluorescence protocols

    Click here to view the general protocols

    Datasheets and documents

    • SDS download

    • Datasheet download

      Download

    Certificate of Compliance

    To download a Certificate of Compliance, please enter your Lot number below:

    References (1)

    Publishing research using ab188401? Please let us know so that we can cite the reference in this datasheet.

    ab188401 has been referenced in 1 publication.

    • Zhan X  et al. Glioma stem-like cells evade interferon suppression through MBD3/NuRD complex-mediated STAT1 downregulation. J Exp Med 217:N/A (2020). PubMed: 32181805

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