Anti-Histone H3 (di methyl K9, tri methyl K9) antibody [6F12-H4] - ChIP Grade (ab71604)

Overview

  • Product nameAnti-Histone H3 (di methyl K9, tri methyl K9) antibody [6F12-H4] - ChIP Grade
    See all Histone H3 primary antibodies
  • Description
    Mouse monoclonal [6F12-H4] to Histone H3 (di methyl K9, tri methyl K9) - ChIP Grade
  • SpecificityIn Western blot ab71604 is blocked by tri methyl K9 and di methyl K9 peptides. It is not blocked by non-modified peptides or by methylated K4 or K27 peptides. Previous batches of ab71604 have generated a ChIP profile consistant with a Histone H3(tri methyl K9) antibody whereas our latest batch displays a profile more consistant with a Histone H3(di methyl K9) antibody. Please contact Scientific Support for further information.
  • Tested applicationsFlow Cyt, WB, ICC/IF, IHC-P, ELISAmore details
  • Species reactivity
    Reacts with: Cow, Human
    Predicted to work with: Mouse, Rat, Rabbit, Hamster, Pig, Saccharomyces cerevisiae, Fruit fly (Drosophila melanogaster), Non Human Primates, a wide range of other species
  • Immunogen

    Synthetic peptide of Human Histone H3, tri methylated at K9.

  • Positive control
    • This antibody gave a positive signal in a calf thymus histone preparation. This antibody gave a positive result in IHC in the following FFPE tissue: Human normal skin.

Properties

Applications

Our Abpromise guarantee covers the use of ab71604 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
Flow Cyt Use 1µg for 106 cells.
WB Use a concentration of 1 µg/ml. Detects a band of approximately 17 kDa (predicted molecular weight: 15 kDa).Can be blocked with Human Histone H3 (tri methyl K9) peptide (ab1773).
ICC/IF Use a concentration of 1 - 5 µg/ml.
IHC-P Use a concentration of 1 µg/ml.
ELISA Use at an assay dependent dilution.

Target

  • FunctionCore 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 similaritiesBelongs to the histone H3 family.
  • Developmental stageExpressed during S phase, then expression strongly decreases as cell division slows down during the process of differentiation.
  • 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.
    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 localizationNucleus. Chromosome.
  • Information by UniProt
  • Database links
  • Alternative names
    • H3 histone family, member J antibody
    • FLJ92264 antibody
    • H3 histone antibody
    • H3 histone family, member A antibody
    • H3 histone family, member B antibody
    • H3 histone family, member C antibody
    • H3 histone family, member D antibody
    • H3 histone family, member F antibody
    • H3 histone family, member H antibody
    • H3 histone family, member I antibody
    • H3 histone family, member K antibody
    • H3 histone family, member L antibody
    • H3 histone, family 3A antibody
    • H3.3A antibody
    • H3/a antibody
    • H3/b antibody
    • H3/c antibody
    • H3/d antibody
    • h3/f antibody
    • H3/h antibody
    • H3/i antibody
    • H3/j antibody
    • H3/k antibody
    • H3/l antibody
    • H31_HUMAN antibody
    • H3F1K antibody
    • H3F3 antibody
    • H3FA antibody
    • H3FB antibody
    • H3FC antibody
    • H3FD antibody
    • H3FF antibody
    • H3FH antibody
    • H3FI antibody
    • H3FJ antibody
    • H3FK antibody
    • H3FL 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 1, H3b antibody
    • Histone 1, H3c antibody
    • Histone 1, H3d antibody
    • Histone 1, H3e antibody
    • Histone 1, H3f antibody
    • Histone 1, H3g antibody
    • Histone 1, H3h antibody
    • Histone 1, H3i antibody
    • Histone 1, H3j antibody
    • Histone cluster 1, H3a antibody
    • Histone cluster 1, H3b antibody
    • Histone cluster 1, H3c antibody
    • Histone cluster 1, H3d antibody
    • Histone cluster 1, H3e antibody
    • Histone cluster 1, H3f antibody
    • Histone cluster 1, H3g antibody
    • Histone cluster 1, H3i antibody
    • Histone cluster 1, H3j antibody
    • Histone H 3 antibody
    • Histone H3.1 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

Anti-Histone H3 (di methyl K9, tri methyl K9) antibody [6F12-H4] - ChIP Grade images

  • All lanes : Anti-Histone H3 (di methyl K9, tri methyl K9) antibody [6F12-H4] - ChIP Grade (ab71604) at 1 µg/ml

    Lane 1 : Calf Thymus Histone Preparation Nuclear Lysate (ab121)
    Lane 2 : Calf Thymus Histone Preparation Nuclear Lysate (ab121) with Human Histone H3 (unmodified ) peptide (ab7228) at 2 µg/ml
    Lane 3 : Calf Thymus Histone Preparation Nuclear Lysate (ab121) with Human Histone H3 (mono methyl K4) peptide (ab1340) at 2 µg/ml
    Lane 4 : Calf Thymus Histone Preparation Nuclear Lysate (ab121) with Histone H3 (di methyl K4) peptide (ab7768) at 2 µg/ml
    Lane 5 : Calf Thymus Histone Preparation Nuclear Lysate (ab121) with Human Histone H3 (tri methyl K4) peptide (ab1342) at 2 µg/ml
    Lane 6 : Calf Thymus Histone Preparation Nuclear Lysate (ab121) with Human Histone H3 (mono methyl K9) peptide (ab1771) at 2 µg/ml
    Lane 7 : Calf Thymus Histone Preparation Nuclear Lysate (ab121) with Human Histone H3 (di methyl K9) peptide (ab1772) at 2 µg/ml
    Lane 8 : Calf Thymus Histone Preparation Nuclear Lysate (ab121) with Human Histone H3 (tri methyl K9) peptide (ab1773) at 2 µg/ml
    Lane 9 : Calf Thymus Histone Preparation Nuclear Lysate (ab121) with Histone H3 (mono methyl K27) peptide (ab1780) at 2 µg/ml
    Lane 10 : Calf Thymus Histone Preparation Nuclear Lysate (ab121) with Human Histone H3 (di methyl K27) peptide (ab1781) at 2 µg/ml
    Lane 11 : Calf Thymus Histone Preparation Nuclear Lysate (ab121) with Human Histone H3 (tri methyl K27) peptide (ab1782) at 2 µg/ml

    Lysates/proteins at 0.5 µg per lane.

    Secondary
    Goat Anti-Mouse IgG H&L (HRP) preadsorbed (ab97040) at 1/10000 dilution
    developed using the ECL technique

    Performed under reducing conditions.

    Predicted band size : 15 kDa


    Exposure time : 3 minutes
  • ICC/IF image of ab71604 stained HeLa cells. The cells were 4% PFA fixed (10 min) and then incubated in 1%BSA / 10% normal Goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab71604, 1µg/ml) overnight at +4°C. The secondary antibody (green) was Alexa Fluor® 488 Goat anti-Mouse IgG (H+L) used at a 1/1000 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (red) at a 1/200 dilution for 1h. DAPI was used to stain the cell nuclei (blue). This antibody also gave a positive result in 4% PFA fixed (10 min) Hek293, MCF7 and HepG2 cells at 5µg/ml, and in 100% Methanol fixed (5 min) HeLa, Hek293, HepG2 and MCF7 cells at 5µg/ml.
  • IHC image of Histone H3 (tri methyl K9, di methyl K9) staining in Human normal skin formalin fixed paraffin embedded tissue section, performed on a Leica BondTM system using the standard protocol F. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20 mins. The section was then incubated with ab71604, 1µg/ml, for 15 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX.

    For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.

  • Overlay histogram showing HeLa cells stained with ab71604 (red line). The cells were fixed with 80% methanol (5 min) and then permeabilized with 0.1% PBS-Tween for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (ab71604, 1µg/1x106 cells) for 30 min at 22ºC. The secondary antibody used was DyLight® 488 goat anti-mouse IgG (H+L) (ab96879) at 1/500 dilution for 30 min at 22ºC. Isotype control antibody (black line) was mouse IgG1 [ICIGG1] (ab91353, 2µg/1x106 cells) used under the same conditions. Acquisition of >5,000 events was performed.

  • All batches of ab71604 are tested in Peptide Array against peptides to different Histone H3 modifications at 2µg/ml. Six dilutions of each peptide are printed on to the Peptide Array in triplicate and results are averaged before being plotted on to a graph. Results show strong binding to Histone H3 - tri methyl K9 peptide (ab1773) and di methyl K9 peptide (ab1772), indicating that this antibody recognises both modifications. Partial cross reactivity is observed with mono methyl K9 peptide (ab1771)

    1. ab1340 - Histone H3 - mono methyl K4
    2. ab7768 - Histone H3 - di methyl K4
    3. ab1342 - Histone H3 - tri methyl K4
    4. ab1771 - Histone H3 - mono methyl K9
    5. ab1772 - Histone H3 - di methyl K9
    6. ab1773 - Histone H3 - tri methyl K9
    7. ab1780 - Histone H3 - mono methyl K27
    8. ab1781 - Histone H3 - di methyl K27
    9. ab1782 - Histone H3 - tri methyl K27
    10. ab7228 - Histone H3 - unmodified

References for Anti-Histone H3 (di methyl K9, tri methyl K9) antibody [6F12-H4] - ChIP Grade (ab71604)

This product has been referenced in:
  • Verver DE  et al. Role for rodent Smc6 in pericentromeric heterochromatin domains during spermatogonial differentiation and meiosis. Cell Death Dis 4:e749 (2013). IHC-P ; Mouse . Read more (PubMed: 23907463) »
  • Yajima H  et al. The complexity of DNA double strand breaks is a critical factor enhancing end-resection. DNA Repair (Amst) 12:936-46 (2013). ICC/IF ; Mouse, Human . Read more (PubMed: 24041488) »

See all 2 Publications for this product

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I can confirm that this antibody (ab71604: Anti-Histone H3 (tri methyl K9, di methyl K9) antibody [6F12-H4] - ChIP Grade) has not been used in any plant species; it has been characterized cow and human samples. Read More

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