Anti-Histone H3 (tri methyl K27) antibody [mAbcam 6147] (ab6147)

Overview

  • Product nameAnti-Histone H3 (tri methyl K27) antibody [mAbcam 6147]
    See all Histone H3 primary antibodies
  • Description
    Mouse monoclonal [mAbcam 6147] to Histone H3 (tri methyl K27)
  • SpecificityBy Western blot, this antibody is blocked strongly by tri methyl K27 peptide and weakly by di methyl K27 peptide. By ELISA, it is specific for tri methyl K27 peptide as shown (although it also binds very weakly with di methyl K27 peptide). The sequence which it reacts is found in all Mammals and a wide range of other species, including D. melanogaster, Arabidopsis, Chicken and Xenopus. The antibody will react with any of the above species where the modification is present. Reactivity is not certain in S. pombe and S. cerevisiae as the equivalent protein sequence differs slightly from species listed above.
  • Tested applicationsICC/IF, IHC-P, ELISA, WB, Flow Cytmore details
  • Species reactivity
    Reacts with: Mouse, Cow, Human, Caenorhabditis elegans
    Predicted to work with: Rat, Chicken, Xenopus laevis, Arabidopsis thaliana, Fruit fly (Drosophila melanogaster)

    Does not react with

    Carp
  • Immunogen

    Synthetic peptide derived from residues 1 - 100 of Human Histone H3, tri methylated at K27.

    . Hybridomas were prepared and the resulting clones were positively screened by ELISA against the immunising peptide. Clones were negatively screened against both the corresponding unmodified peptide and also against a peptide corresponding to tri methylated K9 of Histone H3.

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
    Constituents: PBS, pH 7.4
  • Concentration information loading...
  • PurityIgG fraction
  • Clonality Monoclonal
  • Clone numbermAbcam 6147
  • IsotypeIgG1
  • Light chain typekappa
  • Research Areas

Applications

Our Abpromise guarantee covers the use of ab6147 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 Use a concentration of 1 - 5 µg/ml.
IHC-P Use at an assay dependent concentration.
ELISA Use a concentration of 1 µg/ml. PubMed: 17641388
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 K27) peptide (ab1782).
Flow Cyt Use 1-2µg for 106 cells.

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 K27) antibody [mAbcam 6147] images

  • ICC/IF image of ab6147 stained mouse 2 cell embryo. Cells were fixed with formaldehyde,  permeabilized with 0.5% Triton, and incubated with ab6147 at 1/50 for 2 hours at 37°C. Blocking was performed in 5% FCS serum for 30 minutes at 37°C. The secondary antibody (green) was Alexa Fluor® 488 goat anti-mouse IgG used at a 1/250 dilution. PI was used to stain the DNA (Red).

    See Abreview

  • All lanes : Anti-Histone H3 (tri methyl K27) antibody [mAbcam 6147] (ab6147) at 5 µg/ml

    Lane 1 : Calf thymus histone lysate
    Lane 2 : Calf thymus histone lysate with Human Histone H3 (unmodified ) peptide (ab2623) at 1 µg/ml
    Lane 3 : Calf thymus histone lysate with Human Histone H3 (mono methyl K4) peptide (ab1340) at 1 µg/ml
    Lane 4 : Calf thymus histone lysate with Histone H3 (di methyl K4) peptide (ab7768) at 1 µg/ml
    Lane 5 : Calf thymus histone lysate with Human Histone H3 (tri methyl K4) peptide (ab1342) at 1 µg/ml
    Lane 6 : Calf thymus histone lysate with Human Histone H3 (mono methyl K9) peptide (ab1771) at 1 µg/ml
    Lane 7 : Calf thymus histone lysate with Human Histone H3 (di methyl K9) peptide (ab1772) at 1 µg/ml
    Lane 8 : Calf thymus histone lysate with Human Histone H3 (tri methyl K9) peptide (ab1773) at 1 µg/ml
    Lane 9 : Calf thymus histone lysate with Histone H3 (mono methyl K27) peptide (ab1780) at 1 µg/ml
    Lane 10 : Calf thymus histone lysate with Human Histone H3 (di methyl K27) peptide (ab1781) at 1 µg/ml
    Lane 11 : Calf thymus histone lysate with Human Histone H3 (tri methyl K27) peptide (ab1782) at 1 µg/ml

    Secondary
    Goat Anti-Mouse IgG H&L (HRP) (ab6789) at 1/5000 dilution
    developed using the ECL technique

    Performed under reducing conditions.

    Predicted band size : 15 kDa


    Exposure time : 2 minutes
  • ELISA using ab6147.

    The red line indicates binding to the tri methyl K27 peptide (ab1782). Weak binding (brown line) was seen to the di methyl K27 peptide (ab1781). Binding to the following peptides was not seen:
    Unmodified K27 (ab2623),
    mono methyl K27 (ab1780),
    mono methyl K9 (ab1771),
    di methyl K9 (ab1772),
    tri methyl K9 (ab1773),
    mono methyl K4 (ab1340),
    di methyl K4 (ab7768),
    tri methyl K4 (ab1342).

    This indicates the specificity of ab6147 for tri methyl K27 of Histone H3.

  • ICC/IF image of ab6147 stained human HeLa cells. The cells were PFA fixed (10 min), permabilised in TBS-T (20 min) and incubated with the antibody (ab6147, 1µg/ml) for 1h at room temperature. 1%BSA / 10% normal goat serum / 0.3M glycine was used to quench autofluorescence and block non-specific protein-protein interactions. 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). DAPI was used to stain the cell nuclei (blue).

  • Overlay histogram showing HeLa cells stained with ab6147 (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 (ab6147, 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. This antibody gave a positive signal in HeLa cells fixed with 4% paraformaldehyde (10 min)/permeabilized with 0.1% PBS-Tween for 20 min used under the same conditions.

  • Immunofluorescence analysis of C. elegans germline cells and gonads, staining Histone H3 (tri methyl K27) with ab6147.

    Cells were fixed with methanol, frozen and cracked in liquid nitrogen and blocked with 0.5% BSA for 1 hour at 20°C. Samples were incubated with primary antibody (1/500 in diluent) for 14 hours at 4°C. An FITC-conjugated donkey anti-mouse polyclonal IgG (1/300) was used as the secondary antibody.

    See Abreview

References for Anti-Histone H3 (tri methyl K27) antibody [mAbcam 6147] (ab6147)

This product has been referenced in:
  • Peach SE  et al. Quantitative assessment of ChIP-grade antibodies directed against histone modifications reveals patterns of co-occurring marks on histone protein molecules. Mol Cell Proteomics : (2012). Read more (PubMed: 22442256) »
  • Cornacchia D  et al. Mouse Rif1 is a key regulator of the replication-timing programme in mammalian cells. EMBO J 31:3678-90 (2012). ICC/IF . Read more (PubMed: 22850673) »

See all 4 Publications for this product

Product Wall

Application Immunocytochemistry/ Immunofluorescence
Blocking step BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 3% · Temperature: 21°C
Sample Human Cell (U2OS)
Specification U2OS
Permeabilization Yes - NP40
Fixative Formaldehyde
Username

Abcam user community

Verified customer

Submitted Jul 04 2014

Abcam guarantees this product to work in the species/application used in this Abreview.
Application Immunocytochemistry/ Immunofluorescence
Blocking step Serum as blocking agent for 30 minute(s) · Concentration: 5% · Temperature: 37°C
Sample Mouse Cell (2 cell embryo)
Specification 2 cell embryo
Permeabilization Yes - 0.5% Triton
Fixative Formaldehyde
Username

Dr. Maki Asami

Verified customer

Submitted May 15 2013

Abcam guarantees this product to work in the species/application used in this Abreview.
Application Immunocytochemistry/ Immunofluorescence
Sample Caenorhabditis elegans Cell (germ cell and embryo)
Specification germ cell and embryo
Fixative Methanol
Permeabilization Yes - freeze and crack in liquid nitrogen
Blocking step BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 0.5% · Temperature: 20°C
Username

Carlos Carvalho

Verified customer

Submitted Jan 18 2013


I am very pleased to hear you would like to accept our offer and test ab6147 in c. elegans. This code will give you $XXX off your next order before the expiration date. To redeem this offer, please submit an Abreview for c. elegans and include thi...

Read More

Thank you very much for your interest our AbTrial program.

To our knowledge, ab6147 and ab7766 have not been tested in c. elegans. However by participating in our AbTrial program you can now use our products in an untested application or speci...

Read More

Vielen Dank für Ihren Anruf.

Es tut mir sehrleid, dass Sie Probleme auch mit diesem Antikörper hatten. Wie am Telefon besprochen, werden wir Ihnen diesen Antikörper gerne ersetzen - entweder mit einer Gutschrift, Rückerst...

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 Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)
Sample Mouse Tissue sections (brain)
Specification brain
Fixative Methanol
Antigen retrieval step Heat mediated - Buffer/Enzyme Used: Citrate buffer
Permeabilization Yes - 0.5% Triton X100
Blocking step Serum as blocking agent for 2 hour(s) and 0 minute(s) · Concentration: 15% · Temperature: 25°C
Username

Dr. Chantal Desmaze

Verified customer

Submitted Dec 07 2011

Code de remise : ******* (ab6147 testé en IHC-P) Date d’expiration : JJ MM AAAA Ce code de réduction est utilisable sous 4 mois et pour l’achat d’un autre anticorps primaire. Vous devez dans un premier temps soumettre une Abreview avec vos résu...

Read More

Thank you for your inquiry. The clone number is actually "mAbcam 6147." We generate this antibody in-house and so have assigned this clone number so that it is easily traced back to it's origins. I hope this information helps. Please co...

Read More

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