Anti-Histone H3 (tri methyl K9) antibody [mAbcam 6001] (ab6001)

Overview

  • Product nameAnti-Histone H3 (tri methyl K9) antibody [mAbcam 6001]
    See all Histone H3 primary antibodies
  • Description
    Mouse monoclonal [mAbcam 6001] to Histone H3 (tri methyl K9)
  • SpecificityBy ELISA ab6001 binds to tri methyl K9 peptide, but not to unmodified aa1-13 peptide, mono methyl K4, di methyl K4, tri methyl K4, mono methyl K9, di methyl K9, di methyl K27 or tri methyl K27 peptide.
  • Tested applicationsWB, ICC/IFmore details
  • Species reactivity
    Reacts with: Mouse, Cow, Human
  • Immunogen

    Synthetic peptide conjugated to KLH derived from within residues 1 - 100 of Human Histone H3.

    (Peptide available as ab1773.) Clones were positively screened by ELISA against the immunising peptide conjugated to BSA. Clones were negatively screened against both the equivalent unmodified peptide conjugated to BSA and also a tri methyl K27 peptide conjugated to BSA.

  • Positive control
    • This antibody gave a positive result when used in the following methanol fixed cell lines: HepG2.

Properties

  • FormLiquid
  • Storage instructionsShipped at 4°C. Store at +4°C short term (1-2 weeks). Upon delivery aliquot. Store at -20°C or -80°C. Avoid freeze / thaw cycle.
  • Storage bufferPreservative: 0.02% Sodium azide
    Constituent: PBS
  • Concentration information loading...
  • PurityIgG fraction
  • Clonality Monoclonal
  • Clone numbermAbcam 6001
  • IsotypeIgG2a
  • Light chain typekappa
  • Research Areas

Applications

Our Abpromise guarantee covers the use of ab6001 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 Use a concentration of 1 - 5 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 5 µg/ml.
  • Application notesIs unsuitable for ChIP.
  • 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 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
      • 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
      • 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 (tri methyl K9) antibody [mAbcam 6001] images

    • ICC/IF image of ab6001 stained HepG2 cells. The cells were 100% methanol fixed (5 min) and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Triton for 1h to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody ab6001 at 5µg/ml overnight at +4°C. The secondary antibody (green) was a goat anti-mouse DyLight® 488 (ab96879) IgG (H+L) used at a 1/250 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) at a concentration of 1.43µM.

    • All batches of ab6001 are tested in Peptide Array against peptides to different Histone H3 modifications at 20µg/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), indicating that this antibody specifically recognises the Histone H3 - tri methyl K9 modification.

      1. ab1773 - Histone H3 - tri methyl K9
      2. ab1340 - Histone H3 - mono methyl K4
      3. ab7768 - Histone H3 - di methyl K4
      4. ab1342 - Histone H3 - tri methyl K4
      5. ab1771 - Histone H3 - mono methyl K9
      6. ab1772 - Histone H3 - di 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
    • Anti-Histone H3 (tri methyl K9) antibody [mAbcam 6001] (ab6001) at 3 µg/ml + Calf thymus histone lysate

      Secondary
      Rabbit Anti-Mouse IgG H&L (HRP) (ab6728) at 1/5000 dilution
      developed using the ECL technique

      Performed under reducing conditions.

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


      Exposure time : 5 minutes
    • SK-N-SH cells fixed in 4% paraformaldehyde, permeabilized in 0.5% Triton X-100 and incubated for 1 hour with ab6001 (1/200 dilution). ab6001 staining is localized to the nucleus (green). The cells were counterstained with DAPI (blue). 100x magnification.
    • ab6001 (1/200) staining Histone H3 (tri methyl K9) in assynchronous HeLa cells (green). Cells were fixed in formaldehyde, permeabilized with 0.5% Triton X100/PBS and counterstained with DAPI in order to highlight the nucleus (red). For further experimental details please see Abreview.

      See Abreview

    References for Anti-Histone H3 (tri methyl K9) antibody [mAbcam 6001] (ab6001)

    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) »
    • Youn MY  et al. JMJD5, a Jumonji C (JmjC) Domain-containing Protein, Negatively Regulates Osteoclastogenesis by Facilitating NFATc1 Protein Degradation. J Biol Chem 287:12994-3004 (2012). WB . Read more (PubMed: 22375008) »

    See all 7 Publications for this product

    Product Wall

    Application Immunocytochemistry/ Immunofluorescence
    Blocking step BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: RT°C
    Sample Human Cell (preimplantation embryo)
    Specification preimplantation embryo
    Permeabilization Yes - 0.25% TritonX-100
    Fixative Paraformaldehyde
    Username

    Abcam user community

    Verified customer

    Submitted May 13 2014

    Abcam guarantees this product to work in the species/application used in this Abreview.
    Application Immunocytochemistry/ Immunofluorescence
    Sample Mouse Cell (Oocyte)
    Specification Oocyte
    Fixative Formaldehyde
    Permeabilization Yes - 0.25% Triton
    Blocking step Serum as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 3% · Temperature: 37°C
    Username

    Dr. Maki Asami

    Verified customer

    Submitted Oct 03 2012

    Thank you very much for your email and for letting us know about the trouble with ab6001.

    Would you mind answering a few questions, just so I better understand the situation?

    1) What kind of tissue are you staining (paraffin-embedde...

    Read More
    Abcam guarantees this product to work in the species/application used in this Abreview.
    Application Western blot
    Sample Human Cell lysate - whole cell (U2OS)
    Loading amount 25 µg
    Specification U2OS
    Gel Running Conditions Reduced Denaturing
    Blocking step Milk as blocking agent for 2 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 21°C
    Username

    Abcam user community

    Verified customer

    Submitted Aug 01 2012

    Thank you very much for contacting us with your question and for your patience.

    I aligned the immunogen of ab6001 against the S. purpuratus histone H3 protein sequence, and there is 100% identity between the sequences. So, I would predict the...

    Read More

    Thank you for contacting Abcam.

    All of our products are for research purposes only and are not certified for clinical research applications.

    I am sorry that I could not be more helpful in this matter.

    Abcam guarantees this product to work in the species/application used in this Abreview.
    Application Immunocytochemistry/ Immunofluorescence
    Sample Human Cell (HeLa)
    Specification HeLa
    Fixative Formaldehyde
    Permeabilization Yes - 0.5% Triton-X100 in PBS
    Username

    Dr. Kirk McManus

    Verified customer

    Submitted Feb 02 2012

    Abcam guarantees this product to work in the species/application used in this Abreview.
    Application Immunocytochemistry/ Immunofluorescence
    Sample Human Cell (HeLa)
    Specification HeLa
    Fixative Paraformaldehyde
    Permeabilization Yes - 0.5% Triton X100 in PBS
    Username

    Dr. Kirk McManus

    Verified customer

    Submitted Oct 14 2008

    Abcam guarantees this product to work in the species/application used in this Abreview.
    Application Immunocytochemistry
    Sample Cow Cell (bovine fetal fibroblast)
    Specification bovine fetal fibroblast
    Fixative Paraformaldehyde
    Blocking step BSA as blocking agent for 1 hour(s) and 30 minute(s) · Concentration: 3%
    Username

    Abcam user community

    Verified customer

    Submitted Jun 17 2008

    Thank you for your enquiry. To answer your questions: 1) EZH2 antibody ab3748. This antibody was tested and shown by both our in house chromatin post doc and a collaborator to be unsuitable for chromatin IP. Unfortunately "unsuitable for ChIP" was ...

    Read More

    Please note: All products are "FOR RESEARCH USE ONLY AND ARE NOT INTENDED FOR DIAGNOSTIC OR THERAPEUTIC USE"