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 (methylated) 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 Cyt more details
• Species reactivity
  Reacts with: Mouse, Cow, Human
  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.

  Read Abcam's proprietary immunogen policy

  . 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 instructionsStore at +4°C short term (1-2 weeks). Aliquot and store at -20°C or -80°C. Avoid repeated freeze / thaw cycles.
• 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
  • Epigenetics and Nuclear Signaling
  • Histones
  • H3
  • Methylated

Applications

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
  modificationsAcetylation 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.

Target information above from: UniProt accession P68431 The UniProt Consortium
The Universal Protein Resource (UniProt) in 2010
Nucleic Acids Res. 38:D142-D148 (2010) .

Information by UniProt
• Database links
  • Entrez Gene: 427887 Chicken
  • Entrez Gene: 326601 Cow
  • Entrez Gene: 8350 Human
  • Entrez Gene: 8351 Human
  • Entrez Gene: 8352 Human
  • Entrez Gene: 8353 Human
  • Entrez Gene: 8354 Human
  • Entrez Gene: 8355 Human

  • Entrez Gene: 8356 Human
  • Entrez Gene: 8357 Human
  • Entrez Gene: 8358 Human
  • Entrez Gene: 8968 Human
  • Entrez Gene: 319152 Mouse
  • Entrez Gene: 319153 Mouse
  • Entrez Gene: 360198 Mouse
  • Entrez Gene: 97908 Mouse
  • Entrez Gene: 100364501 Rat
  • Entrez Gene: 100365669 Rat
  • Entrez Gene: 291159 Rat
  • Entrez Gene: 679994 Rat
  • Entrez Gene: 680511 Rat
  • Entrez Gene: 682330 Rat
  • Omim: 601128 Human
  • SwissProt: P59226 Arabidopsis thaliana
  • SwissProt: P84229 Chicken
  • SwissProt: P68432 Cow
  • SwissProt: P02299 Fruit fly (Drosophila melanogaster)
  • SwissProt: Q93081 Human
  • SwissProt: Q16695 Human
  • SwissProt: P68431 Human
  • SwissProt: P68433 Mouse
  • SwissProt: Q6LED0 Rat
  • Unigene: 132854 Human
  • Unigene: 247813 Human
  • Unigene: 247814 Human
  • Unigene: 248176 Human
  • Unigene: 443021 Human
  • Unigene: 484990 Human
  • Unigene: 532144 Human
  • Unigene: 533292 Human
  • Unigene: 546315 Human
  • Unigene: 586261 Human
  • Unigene: 591778 Human
  • Unigene: 221301 Mouse
  • Unigene: 261657 Mouse
  • Unigene: 377874 Mouse
  • Unigene: 390558 Mouse
  • Unigene: 397328 Mouse
  • Unigene: 138090 Rat

  see all

• Alternative names
  FLJ92264 antibodyH3 histone antibodyH3 histone antibody

  H3 histone family, member A antibodyH3/a antibodyH3/b antibodyH3/c antibodyH3/d antibodyh3/f antibodyH3/h antibodyH3/i antibodyH3/j antibodyH3/k antibodyH3/l antibodyH31_HUMAN antibodyH3F1K antibodyH3F3 antibodyH3F3 antibodyH3FA antibodyH3FB antibodyH3FC antibodyH3FD antibodyH3FF antibodyH3FH antibodyH3FI antibodyH3FJ antibodyH3FK antibodyH3FL antibodyHIST1H3A antibodyHIST1H3B antibodyHIST1H3C antibodyHIST1H3D antibodyHIST1H3E antibodyHIST1H3F antibodyHIST1H3G antibodyHIST1H3H antibodyHIST1H3I antibodyHIST1H3J antibodyHIST3H3 antibodyHIST3H3 antibodyHistone 1, H3a antibodyHistone cluster 1, H3a antibodyHistone cluster 1, H3b antibodyHistone cluster 1, H3c antibodyHistone cluster 1, H3d antibodyHistone cluster 1, H3e antibodyHistone cluster 1, H3f antibodyHistone cluster 1, H3g antibodyHistone cluster 1, H3i antibodyHistone cluster 1, H3j antibodyHistone H 3 antibodyHistone H3.1 antibodyHistone H3.1 antibodyHistone H3/a antibodyHistone H3/b antibodyHistone H3/c antibodyHistone H3/d antibodyHistone H3/f antibodyHistone H3/h antibodyHistone H3/i antibodyHistone H3/j antibodyHistone H3/k antibodyHistone H3/l antibody

  see all

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

• 

  Immunocytochemistry/ Immunofluorescence - Anti-Histone H3 (tri methyl K27) antibody [mAbcam 6147] (ab6147)Image courtesy of Maki Asami, University of Bath

  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).

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• 

  Western blot - Histone H3 (tri methyl K27) antibody [mAbcam 6147] (ab6147)
  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 Histone H3 peptide - unmodified (ab2623) at 1 µg/ml
  Lane 3 : Calf thymus histone lysate with Histone H3 peptide - mono methyl K4 (ab1340) at 1 µg/ml
  Lane 4 : Calf thymus histone lysate with Histone H3 peptide - di methyl K4 (ab7768) at 1 µg/ml
  Lane 5 : Calf thymus histone lysate with Histone H3 peptide - tri methyl K4 (ab1342) at 1 µg/ml
  Lane 6 : Calf thymus histone lysate with Histone H3 peptide - mono methyl K9 (ab1771) at 1 µg/ml
  Lane 7 : Calf thymus histone lysate with Histone H3 peptide - di methyl K9 (ab1772) at 1 µg/ml
  Lane 8 : Calf thymus histone lysate with Histone H3 peptide - tri methyl K9 (ab1773) at 1 µg/ml
  Lane 9 : Calf thymus histone lysate with Histone H3 peptide - mono methyl K27 (ab1780) at 1 µg/ml
  Lane 10 : Calf thymus histone lysate with Histone H3 peptide - di methyl K27 (ab1781) at 1 µg/ml
  Lane 11 : Calf thymus histone lysate with Histone H3 peptide - tri methyl K27 (ab1782) at 1 µg/ml
  
  Secondary
  Goat polyclonal Secondary Antibody to 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 - Histone H3 (tri methyl K27) antibody [mAbcam 6147] (ab6147)

  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.

• 

  Immunocytochemistry/ Immunofluorescence - Histone H3 (tri methyl K27) antibody [mAbcam 6147] (ab6147)
  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).

• 

  Flow Cytometry-Anti-Histone H3 (tri methyl K27) antibody [mAbcam 6147](ab6147)
  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.
  
  
• 

  Immunocytochemistry/ Immunofluorescence - Anti-Histone H3 (tri methyl K27) antibody [mAbcam 6147] (ab6147)This image is courtesy of an Abreview submitted by Carlos Carvalho
  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.

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