Recombinant Anti-Histone H3 (di methyl K9) antibody [Y49] - ChIP Grade (ab32521)


  • Product name

    Anti-Histone H3 (di methyl K9) antibody [Y49] - ChIP Grade
    See all Histone H3 primary antibodies
  • Description

    Rabbit monoclonal [Y49] to Histone H3 (di methyl K9) - ChIP Grade
  • Host species

  • Specificity

    The antibody only detects Histone H3 dimethylated on Lysine 9.

  • Tested applications

    Suitable for: ICC/IF, WB, Flow Cyt, ChIPmore details
    Unsuitable for: IHC or IP
  • Species reactivity

    Reacts with: Mouse, Rat, Human
  • Immunogen

    Synthetic peptide within Human Histone H3 aa 1-100 (di methyl K9). The exact sequence is proprietary.

  • Positive control

    • WB: HeLa cell lysate. ICC/IF: HeLa cells. Flow Cyt: HeLa cells. ChIP: Chromatin prepared from HeLa cells.
  • General notes

    A trial size is available to purchase for this antibody.


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

    This product is a recombinant rabbit monoclonal antibody.



Our Abpromise guarantee covers the use of ab32521 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
ICC/IF 1/500.
WB 1/1000. Detects a band of approximately 17 kDa (predicted molecular weight: 15 kDa).
Flow Cyt 1/220.
ChIP Use 5 µg for 25 µg of chromatin.
  • Application notes
    Is unsuitable for IHC or IP.
  • Target

    • Function

      Core component of nucleosome. 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 during S phase, then expression strongly decreases as cell division slows down during the process of differentiation.
    • Post-translational

      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.
      Methylation at Lys-5 (H3K4me), Lys-37 (H3K36me) and Lys-80 (H3K79me) are linked to gene activation. 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) are linked to gene repression. 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.
      Monoubiquitinated by RAG1 in lymphoid cells, monoubiquitination is required for V(D)J recombination (By similarity). Ubiquitinated by the CUL4-DDB-RBX1 complex in response to ultraviolet irradiation. This may weaken the interaction between histones and DNA and facilitate DNA accessibility to repair proteins.
    • Cellular localization

      Nucleus. Chromosome.
    • Information by UniProt
    • Database links

    • Alternative names

      • H3 histone family member E pseudogene antibody
      • H3 histone family, member A antibody
      • H3/A antibody
      • H31_HUMAN antibody
      • H3F3 antibody
      • H3FA antibody
      • Hist1h3a antibody
      • HIST1H3B antibody
      • HIST1H3C antibody
      • HIST1H3D antibody
      • HIST1H3E antibody
      • HIST1H3F antibody
      • HIST1H3G antibody
      • HIST1H3H antibody
      • HIST1H3I antibody
      • HIST1H3J antibody
      • HIST3H3 antibody
      • histone 1, H3a antibody
      • Histone cluster 1, H3a antibody
      • Histone H3 3 pseudogene antibody
      • Histone H3.1 antibody
      • Histone H3/a antibody
      • Histone H3/b antibody
      • Histone H3/c antibody
      • Histone H3/d antibody
      • Histone H3/f antibody
      • Histone H3/h antibody
      • Histone H3/i antibody
      • Histone H3/j antibody
      • Histone H3/k antibody
      • Histone H3/l antibody
      see all


    • Chromatin was prepared from HeLa cells according to the Abcam X-ChIP protocol. Cells were fixed with formaldehyde for 10 minutes. The ChIP was performed with 25µg of chromatin, 5µg of ab32521 (blue), and 20µl of Protein A/G sepharose beads. No antibody was added to the beads control (yellow). 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.

    • All lanes : Anti-Histone H3 (di methyl K9) antibody [Y49] - ChIP Grade (ab32521) at 1/1000 dilution

      Lane 1 : HeLa cell lysate
      Lane 2 : recombinant Histone H3

      Predicted band size: 15 kDa
      Observed band size: 17 kDa
      why is the actual band size different from the predicted?

    • Immunocytochemistry/Immunofluorescence analysis of HeLa (human cervix adenocarcinoma) labelling Histone H3 (di methyl K9) with purified ab32521 at 1/500. 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 (Ab150077). Nuclei counterstained with DAPI (blue).

      Control: PBS only

    • Flow Cytometry analysis of HeLa (human cervix adenocarcinoma) cells labeling Histone H3 (di methyl K9) with purified ab32521 at 1/220 dilution (10ug/mL) (red). Cells were fixed with 4% paraformaldehyde and permeabilised with 90% methanol. A Goat anti rabbit IgG (Alexa Fluor® 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.

    • ab32521 (1/500) staining Histone H3 di-methyl K9 in HeLa cells (green). Cells were fixed in paraformaldehyde, permeabilised with 0.5% Triton X100 and counterstained with DAPI in order to highlight the nucleus (red). For further experimental details, please refer to Abreview.

      See Abreview


    This product has been referenced in:

    See all 7 Publications for this product

    Customer reviews and Q&As

    1-3 of 3 Abreviews or Q&A


    For detecting nuclear proteins via western blot we recommend using a RIPA buffer or cell fractionation protocol in preparation of your cell lysate. I attached our protocol book to this email for information regarding specific cell lysate preparation procedures. Below are the fractionation kits that we talked about during your phone call:
    Nuclear Extraction Kit (ab113474)
    Histone Extraction Kit (ab113476)

    The Histone Extraction Kit can be used to extract histones from as low as 1 million cells or 1 mg of tissue as stated on the datasheet. These lower limits are good guidelines for the Nuclear Extraction Kit as well.

    Since ab1220 (anti-histone H3 di methyl K9) is the only antibody in your provided list that is raised in mouse (the others are raised in rabbit), we discussed substituting the rabbit monoclonal antibody ab32521 ( (anti-histone H3 di methyl K9) so you can use the same secondary for each antibody. Since ab32521 is a rabbit monoclonal antibody, it also qualifies for our RabMAB promotion. Details of this promotion are at the end of this email.

    A list of our anti-rabbit secondary antibodies is given through the link below, depending on the detection method and conjugation that you prefer:[0]=rabbit&X-Requested-With=XMLHttpRequest

    Together these antibodies could be used to detect the presence of histone H3 in your samples as well as the methylation status of Lysine 9. To complete the spectrum, Abcam also sells an anti-histone H3 mono methyl K9 antibody (ab9045, As we discussed, ab3790 (anti-KMT8/Riz1/Riz2) detects the presence of PR domain zinc finger protein 2, which is a histone methyltransferase that specifically methylates Lysine 9 of histone H3 according to SwissProt (, thus explaining its connection to the anti-histone H3 antibodies that detect the methylation status of Lysine 9

    Read More
    Immunocytochemistry/ Immunofluorescence
    Human Cell (HeLa)
    Yes - 0.5% Triton X100

    Dr. Kirk Mcmanus

    Verified customer

    Submitted Sep 07 2011

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