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RecombinantRabMAb

Recombinant Anti-Histone H3.3 (mutated G34W) antibody [EPR23581-39] - ChIP Grade (ab272691)

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ChIP - Anti-Histone H3.3 (mutated G34W) antibody [EPR23581-39] - ChIP Grade (ab272691)
  • Western blot - Anti-Histone H3.3 (mutated G34W) antibody [EPR23581-39] - ChIP Grade (ab272691)
  • Flow Cytometry (Intracellular) - Anti-Histone H3.3 (mutated G34W) antibody [EPR23581-39] - ChIP Grade (ab272691)
  • Immunocytochemistry/ Immunofluorescence - Anti-Histone H3.3 (mutated G34W) antibody [EPR23581-39] - ChIP Grade (ab272691)
  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Histone H3.3 (mutated G34W) antibody [EPR23581-39] - ChIP Grade (ab272691)
  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Histone H3.3 (mutated G34W) antibody [EPR23581-39] - ChIP Grade (ab272691)
  • Dot Blot - Anti-Histone H3.3 (mutated G34W) antibody [EPR23581-39] - ChIP Grade (ab272691)
  • Anti-Histone H3.3 (mutated G34W) antibody [EPR23581-39] - ChIP Grade (ab272691)

Key features and details

  • Produced recombinantly (animal-free) for high batch-to-batch consistency and long term security of supply
  • Rabbit monoclonal [EPR23581-39] to Histone H3.3 (mutated G34W) - ChIP Grade
  • Suitable for: ChIP, Flow Cyt (Intra), WB, ICC/IF, Dot blot, IHC-P
  • Reacts with: Human

Conjugates logo Related conjugates and formulations

Carrier Free

Overview

  • Product name

    Anti-Histone H3.3 (mutated G34W) antibody [EPR23581-39] - ChIP Grade
    See all Histone H3.3 primary antibodies

  • Description

    Rabbit monoclonal [EPR23581-39] to Histone H3.3 (mutated G34W) - ChIP Grade

  • Host species

    Rabbit

  • Tested applications

    Suitable for: ChIP, Flow Cyt (Intra), WB, ICC/IF, Dot blot, IHC-Pmore details

  • Species reactivity

    Reacts with: Human

  • Immunogen

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

  • Positive control

    • WB: HEK-293T transfected with Histone H3.3 G34W expression vector containing a myc-His-tag, whole cell lysate. IHC-P: Human giant tumor of bone. Flow Cyt (intra): 293T cells transfected with myc-tagged Histone H3.3 G34W construct. ICC/IF: 293T cells transfected with Histone H3.3 G34W-Myc plasmid.

  • 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

Applications

The Abpromise guarantee

Our Abpromise guarantee covers the use of ab272691 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
ChIP
Use 2 µg for 25 µg of chromatin.
Flow Cyt (Intra)
1/50.
WB
1/1000. Predicted molecular weight: 15 kDa.
ICC/IF
1/100.
Dot blot
1/1000.
IHC-P
1/250. Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.
Notes
ChIP
Use 2 µg for 25 µg of chromatin.
Flow Cyt (Intra)
1/50.
WB
1/1000. Predicted molecular weight: 15 kDa.
ICC/IF
1/100.
Dot blot
1/1000.
IHC-P
1/250. Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

Target

  • Function

    Variant histone H3 which replaces conventional H3 in a wide range of nucleosomes in active genes. Constitutes the predominant form of histone H3 in non-dividing cells and is incorporated into chromatin independently of DNA synthesis. Deposited at sites of nucleosomal displacement throughout transcribed genes, suggesting that it represents an epigenetic imprint of transcriptionally active chromatin. Nucleosomes wrap and compact DNA into chromatin, limiting DNA accessibility to the cellular machineries which require DNA as a template. Histones thereby play a central role in transcription regulation, DNA repair, DNA replication and chromosomal stability. DNA accessibility is regulated via a complex set of post-translational modifications of histones, also called histone code, and nucleosome remodeling.

  • Sequence similarities

    Belongs to the histone H3 family.

  • Developmental stage

    Expressed throughout the cell cycle independently of DNA synthesis.

  • Post-translational
    modifications

    Acetylation is generally linked to gene activation. Acetylation on Lys-10 (H3K9ac) impairs methylation at Arg-9 (H3R8me2s). Acetylation on Lys-19 (H3K18ac) and Lys-24 (H3K24ac) favors methylation at Arg-18 (H3R17me).
    Citrullination at Arg-9 (H3R8ci) and/or Arg-18 (H3R17ci) by PADI4 impairs methylation and represses transcription.
    Asymmetric dimethylation at Arg-18 (H3R17me2a) by CARM1 is linked to gene activation. Symmetric dimethylation at Arg-9 (H3R8me2s) by PRMT5 is linked to gene repression. Asymmetric dimethylation at Arg-3 (H3R2me2a) by PRMT6 is linked to gene repression and is mutually exclusive with H3 Lys-5 methylation (H3K4me2 and H3K4me3). H3R2me2a is present at the 3' of genes regardless of their transcription state and is enriched on inactive promoters, while it is absent on active promoters.
    Specifically enriched in modifications associated with active chromatin such as methylation at Lys-5 (H3K4me), Lys-37 and Lys-80. Methylation at Lys-5 (H3K4me) facilitates subsequent acetylation of H3 and H4. Methylation at Lys-80 (H3K79me) is associated with DNA double-strand break (DSB) responses and is a specific target for TP53BP1. Methylation at Lys-10 (H3K9me) and Lys-28 (H3K27me), which are linked to gene repression, are underrepresented. Methylation at Lys-10 (H3K9me) is a specific target for HP1 proteins (CBX1, CBX3 and CBX5) and prevents subsequent phosphorylation at Ser-11 (H3S10ph) and acetylation of H3 and H4. Methylation at Lys-5 (H3K4me) and Lys-80 (H3K79me) require preliminary monoubiquitination of H2B at 'Lys-120'. Methylation at Lys-10 (H3K9me) and Lys-28 (H3K27me) are enriched in inactive X chromosome chromatin.
    Phosphorylated at Thr-4 (H3T3ph) by GSG2/haspin during prophase and dephosphorylated during anaphase. Phosphorylation at Ser-11 (H3S10ph) by AURKB is crucial for chromosome condensation and cell-cycle progression during mitosis and meiosis. In addition phosphorylation at Ser-11 (H3S10ph) by RPS6KA4 and RPS6KA5 is important during interphase because it enables the transcription of genes following external stimulation, like mitogens, stress, growth factors or UV irradiation and result in the activation of genes, such as c-fos and c-jun. Phosphorylation at Ser-11 (H3S10ph), which is linked to gene activation, prevents methylation at Lys-10 (H3K9me) but facilitates acetylation of H3 and H4. Phosphorylation at Ser-11 (H3S10ph) by AURKB mediates the dissociation of HP1 proteins (CBX1, CBX3 and CBX5) from heterochromatin. Phosphorylation at Ser-11 (H3S10ph) is also an essential regulatory mechanism for neoplastic cell transformation. Phosphorylated at Ser-29 (H3S28ph) by MLTK isoform 1, RPS6KA5 or AURKB during mitosis or upon ultraviolet B irradiation. Phosphorylation at Thr-7 (H3T6ph) by PRKCBB is a specific tag for epigenetic transcriptional activation that prevents demethylation of Lys-5 (H3K4me) by LSD1/KDM1A. At centromeres, specifically phosphorylated at Thr-12 (H3T11ph) from prophase to early anaphase, by DAPK3 and PKN1. Phosphorylation at Thr-12 (H3T11ph) by PKN1 is a specific tag for epigenetic transcriptional activation that promotes demethylation of Lys-10 (H3K9me) by KDM4C/JMJD2C. Phosphorylation at Tyr-42 (H3Y41ph) by JAK2 promotes exclusion of CBX5 (HP1 alpha) from chromatin. Phosphorylation on Ser-32 (H3S31ph) is specific to regions bordering centromeres in metaphase chromosomes.
    Ubiquitinated. Monoubiquitinated by RAG1 in lymphoid cells, monoubiquitination is required for V(D)J recombination.

  • Cellular localization

    Nucleus. Chromosome.
  • Information by UniProt

  • Database links

    • Alternative names

      • H3 histone family 3A antibody
      • H3 histone family 3B antibody
      • H3 histone, family 3B (H3.3B) antibody
      • H3.3 antibody
      • H3.3A antibody
      • H3.3B antibody
      • H33_HUMAN antibody
      • H3F3 antibody
      • H3F3A antibody
      • H3f3b antibody
      • Histone H3.3 antibody
      • Histone H3.3Q antibody
      • Histone H3.A antibody
      • Histone H3.B antibody
      • MGC87782 antibody
      • MGC87783 antibody
      see all

    Images

    • ChIP - Anti-Histone H3.3 (mutated G34W) antibody [EPR23581-39] - ChIP Grade (ab272691)
      ChIP - Anti-Histone H3.3 (mutated G34W) antibody [EPR23581-39] - ChIP Grade (ab272691)

      Chromatin was prepared from HEK-293T transfected with myc-His tagged Histone H3.3 mutated G34W and Histone H3.3 WT cells according to the Abcam Dual-X-ChIP protocol*. Cells were fixed with formaldehyde for 10min.
      The ChIP was performed with 25 µg of chromatin, 2 µg of ab272691 (red), or 2 µg of rabbit normal IgG ab172730 (gray) and 20 µl of Protein A/G sepharose beads. The immunoprecipitated DNA was quantified by real time PCR (Taqman approach for active and inactive loci, Sybr green approach for heterochromatic loci).
      Primers and probes are located in the first kb of the transcribed region.

      *https://www.abcam.com/resources?keywords=X%20ChIP%20protocol

    • Western blot - Anti-Histone H3.3 (mutated G34W) antibody [EPR23581-39] - ChIP Grade (ab272691)
      Western blot - Anti-Histone H3.3 (mutated G34W) antibody [EPR23581-39] - ChIP Grade (ab272691)
      All lanes : Anti-Histone H3.3 (mutated G34W) antibody [EPR23581-39] - ChIP Grade (ab272691) at 1/1000 dilution

      Lane 1 : HEK-293T (human embryonic kidney) transfected with Histone H3.3 G34W expression vector containing a myc-His-tag®, whole cell lysate
      Lane 2 : HEK-293T transfected with Histone H3.3 (WT) expression vector containing a myc-His-tag®, whole cell lysate

      Lysates/proteins at 40 µg per lane.

      Secondary
      All lanes : Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (ab97051) at 1/50000 dilution

      Predicted band size: 15 kDa
      Observed band size: 20 kDa why is the actual band size different from the predicted?



      Blocking and diluting buffer and concentration: 5% NFDM/TBST.

      Exposure time: 3 minutes.

    • Flow Cytometry (Intracellular) - Anti-Histone H3.3 (mutated G34W) antibody [EPR23581-39] - ChIP Grade (ab272691)
      Flow Cytometry (Intracellular) - Anti-Histone H3.3 (mutated G34W) antibody [EPR23581-39] - ChIP Grade (ab272691)

      Intracellular flow cytometric analysis of 4% paraformaldehyde-fixed 90% methanol-permeabilized HEK-293T (Human embryonic kidney epithelial cell) transfected with myc tagged Histone H3.3 WT construct (Left panel) and myc-tagged Histone H3.3 G34W construct (Right panel) cells labelling Histone H3.3 (mutated G34 W) with ab272691 at 1/50 dilution (1µg). A Goat anti rabbit IgG (Alexa Fluor® 488, ab150077) at 1/2000 dilution was used as the secondary antibody. 

       

       

    • Immunocytochemistry/ Immunofluorescence - Anti-Histone H3.3 (mutated G34W) antibody [EPR23581-39] - ChIP Grade (ab272691)
      Immunocytochemistry/ Immunofluorescence - Anti-Histone H3.3 (mutated G34W) antibody [EPR23581-39] - ChIP Grade (ab272691)

      Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized HEK-293T cells labelling Histone H3.3(mutated G34 W) with ab272691 at 1/100 dilution, followed by ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) antibody at 1/1000 2 µg/ml dilution (Green). Confocal image showing nuclear staining in HEK-293T cells transfected with Histone H3.3 G34W-Myc plasmid, while no staining in HEK-293T cells transfected with H3.3 WT -Myc plasmid. Myc-Tag Mouse mAb (Alexa Fluor® 647) was used to counterstain tubulin at 1/200 dilution (Red). The nuclear counterstain was DAPI (Blue).

      Secondary antibody only control: Secondary antibody is ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) at 1/1000 2 µg/ml dilution.

    • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Histone H3.3 (mutated G34W) antibody [EPR23581-39] - ChIP Grade (ab272691)
      Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Histone H3.3 (mutated G34W) antibody [EPR23581-39] - ChIP Grade (ab272691)

      Immunohistochemical analysis of paraffin-embedded human giant cell tumor of bone tissue labeling Histone H3.3(mutated G34 W) with ab272691 at 1/250 dilution followed by ready to use Goat Anti-Rabbit IgG H&L (HRP). Nuclear staining in human giant cell tumor of bone (PMID: 29757500). Counterstained with Hematoxylin.

      Secondary antibody only control: Secondary antibody is ready to use Goat Anti-Rabbit IgG H&L (HRP).

      Heat mediated antigen retrieval using ab93684 (Tris/EDTA buffer, pH 9.0).

    • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Histone H3.3 (mutated G34W) antibody [EPR23581-39] - ChIP Grade (ab272691)
      Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Histone H3.3 (mutated G34W) antibody [EPR23581-39] - ChIP Grade (ab272691)

      Immunohistochemical analysis of paraffin-embedded human chondroblastoma tissue labeling Histone H3.3(mutated G34 W) with ab272691 at 1/250 dilution followed by ready to use Goat Anti-Rabbit IgG H&L (HRP).

      Negative control: No staining in human chondroblastoma (PMID: 29757500).

      Counterstained with Hematoxylin.

      Secondary antibody only control: Secondary antibody is ready to use Goat Anti-Rabbit IgG H&L (HRP).

      Heat mediated antigen retrieval using ab93684 (Tris/EDTA buffer, pH 9.0).

    • Dot Blot - Anti-Histone H3.3 (mutated G34W) antibody [EPR23581-39] - ChIP Grade (ab272691)
      Dot Blot - Anti-Histone H3.3 (mutated G34W) antibody [EPR23581-39] - ChIP Grade (ab272691)

      Dot blot analysis of Histone H3.3 (mutated G34 W) labeled with ab272691 at 1/1000 dilution.

      Lane 1: Histone H3.3 H3G34W peptide (aa28-37).
      Lane 2: Histone H3.3 H3G34W peptide (aa33-43).
      Lane 3: Histone H3.3 H3G34W peptide (aa28-43).
      Lane 4: Histone H3.3 WT peptide (aa28-43).

      Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution was used as secondary antibody.

      Blocking and dilution buffer: 5% NFDM/TBST.

      Exposure time: 3 minutes.

    • Anti-Histone H3.3 (mutated G34W) antibody [EPR23581-39] - ChIP Grade (ab272691)
      Anti-Histone H3.3 (mutated G34W) antibody [EPR23581-39] - ChIP Grade (ab272691)

    Protocols

    To our knowledge, customised protocols are not required for this product. Please try the standard protocols listed below and let us know how you get on.

    Click here to view the general protocols

    References (0)

    Publishing research using ab272691? Please let us know so that we can cite the reference in this datasheet.

    ab272691 has not yet been referenced specifically in any publications.

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