Anti-Histone H3 (tri methyl K27) antibody [mAbcam 6002] - ChIP Grade (ab6002)

Overview

  • Product nameAnti-Histone H3 (tri methyl K27) antibody [mAbcam 6002] - ChIP Grade
    See all Histone H3 primary antibodies
  • Description
    Mouse monoclonal [mAbcam 6002] to Histone H3 (tri methyl K27) - ChIP Grade
  • SpecificityThis antibody is specific for histone H3 tri-methylated at K27. The sequence is found in all mammals and a wide range of species, including D. melanogaster, Arabidopsis, Chicken and Xenopus. The antibody will react with any of the above species where the modification is present. Reactivity was confirmed using Cow samples. Reactivity is not certain in S. pombe and S. cerevisiae as the equivalent protein sequence differs slightly from the species listed above. A customer reported that the activity against histone H3 in Zea mays (maize) is not good. The antibody is blocked in western blotting by tri methyl K27 peptide and slightly by di methyl K27 peptide. It is not blocked by mono methyl K4, di methyl K4, tri methyl K4, mono methyl K9, di methyl K9, tri methyl K9, mono methyl K27 or unmodified K27 peptides. (See blot below).
  • Tested applicationsIP, ChIP/Chip, ChIP, IHC-P, Flow Cyt, WB, ICC, ICC/IFmore details
  • Species reactivity
    Reacts with: Mouse, Rat, Cow, Human, Arabidopsis thaliana, Fruit fly (Drosophila melanogaster), Plants, Zebrafish, Rhesus monkey, Rice
    Predicted to work with: Rabbit, Chicken, Xenopus laevis, Chinese Hamster
  • 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.

  • General notesNOT SUITABLE for blocking with milk. Block in 5% BSA for 1 hour. Our labs have investigated the blocking conditions for this antibody and found that milk significantly decreases the signal and is therefore not a suitable blocking agent for this antibody (see Western Blot image).

Properties

Applications

Our Abpromise guarantee covers the use of ab6002 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
IP Use at an assay dependent concentration.
ChIP/Chip Use at an assay dependent dilution.
ChIP Use 5-10 µg for 25 µg of chromatin.
IHC-P Use at an assay dependent dilution.
Flow Cyt Use at an assay dependent dilution.
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). Block with BSA. The signal is very weak with milk blocking.
ICC Use at an assay dependent dilution.
ICC/IF Use a concentration of 5 µg/ml.

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 6002] - ChIP Grade images

  • Chromatin was prepared from Hela cells according to the Abcam X-ChIP protocol. Cells were fixed with formaldehyde for 10min. The  ChIP was performed with 25µg of chromatin, 5µg of  ab6002 (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 (tri methyl K27) antibody [mAbcam 6002] - ChIP Grade (ab6002) at 1 µg/ml

    Lane 1 : Calf Thymus Histone Preparation Nuclear Lysate
    Lane 2 : Calf Thymus Histone Preparation Nuclear Lysate with Human Histone H3 peptide (ab17163) at 0.5 µg/ml
    Lane 3 : Calf Thymus Histone Preparation Nuclear Lysate with Human Histone H3 (mono methyl K4) peptide (ab1340) at 0.5 µg/ml
    Lane 4 : Calf Thymus Histone Preparation Nuclear Lysate with Histone H3 (di methyl K4) peptide (ab7768) at 0.5 µg/ml
    Lane 5 : Calf Thymus Histone Preparation Nuclear Lysate with Human Histone H3 (tri methyl K4) peptide (ab1342) at 0.5 µg/ml
    Lane 6 : Calf Thymus Histone Preparation Nuclear Lysate with Human Histone H3 (mono methyl K9) peptide (ab1771) at 0.5 µg/ml
    Lane 7 : Calf Thymus Histone Preparation Nuclear Lysate with Human Histone H3 (di methyl K9) peptide (ab1772) at 0.5 µg/ml
    Lane 8 : Calf Thymus Histone Preparation Nuclear Lysate with Human Histone H3 (tri methyl K9) peptide (ab1773) at 0.5 µg/ml
    Lane 9 : Calf Thymus Histone Preparation Nuclear Lysate with Histone H3 (mono methyl K27) peptide (ab1780) at 0.5 µg/ml
    Lane 10 : Calf Thymus Histone Preparation Nuclear Lysate with Human Histone H3 (di methyl K27) peptide (ab1781) at 0.5 µg/ml
    Lane 11 : Calf Thymus Histone Preparation Nuclear Lysate with Human Histone H3 (tri methyl K27) peptide (ab1782) at 0.5 µg/ml

    Lysates/proteins at 0.5 µg per lane.

    Secondary
    Goat polyclonal to Mouse IgG - H&L - Pre-Adsorbed (HRP) at 1/3000 dilution

    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 : 3 minutes
  • All lanes : Anti-Histone H3 (tri methyl K27) antibody [mAbcam 6002] - ChIP Grade (ab6002) at 1 µg/ml

    Lane 1 : Calf Thymus Histone Preparation Nuclear Lysate at 0.5 µg with BSA BLOCK
    Lane 2 : Calf Thymus Histone Preparation Nuclear Lysate at 0.5 µg with MILK BLOCK
    Lane 3 : MEF1 (Mouse embryonic fibroblast cell line) Whole Cell Lysate at 10 µg with BSA BLOCK
    Lane 4 : NIH 3T3 (Mouse embryonic fibroblast cell line) Whole Cell Lysate at 10 µg with BSA BLOCK
    Lane 5 : MEF1 (Mouse embryonic fibroblast cell line) Whole Cell Lysate at 10 µg with MILK BLOCK
    Lane 6 : NIH 3T3 (Mouse embryonic fibroblast cell line) Whole Cell Lysate at 10 µg with MILK BLOCK

    Secondary
    Goat polyclonal to Mouse IgG - H&L - Pre-Adsorbed (HRP) at 1/3000 dilution

    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 : 12 minutes
  • Immunofluorescent imaging of human cells (U2OS) with ab6002 reveals broadly dispersed interphase nuclear staining corresponding to trimethylation of K27, with multiple foci of brighter staining, exactly agreeing with published studies of K27-trimethyl IF [See figure 3 of Peters et al Mol Cell 12(6):1577-1589 (2003)] .  The lack of nucleolar or cytoplasmic staining background confirms the high specificity of the antibody in this application.   

    IF was performed with a standard paraformaldehyde technique (fixed in PBS buffered PFH 4% for 5 minutes, permeabilised with 0.5% triton-PBS for 5 minutes, blocked with 5% milk / 0.2% tween for one hour.  Primary antibody used at 1/200 in 5% milk / 0.2% TWEEN for one hour, secondary antibody Alexa 488 for 30 minutes.  All blocking and incubation steps carried out at 37 degrees C. 

  • Figure showing the nuclear distribution of H3 (tri-methyl K27) antibody, ab6002 in a) a 46 chromosome, XX cell line, and b) a 49 chromosome, XXXXX cell line.

    The location of facultative heterochromatin at the inactive X chromosome is indicated by white arrow heads.

  • Interphase 10T1/2 mouse fibroblasts were paraformaldehyde fixed (4%), immunofluorescently labeled with anti-trimethyl K27 antibody (ab6002) and counterstained with DAPI.  The merge image presents the DAPI and ab6002 channels as red and green, respectively.  The scale bar represents 3µm.
  • ELISA using ab6002 at antibody concentrations between 1/500 and 1/8000.

    The red line indicates binding to the tri methyl K27 peptide (ab1782). Binding to the following peptides was not seen:
    Unmodified K27 (ab2623),
    mono methyl K27 (ab1780),
    di methyl K27 (ab1781),
    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 ab6002 for tri methyl K27 of Histone H3.

  • ab6002 staining mouse embyronic stem cells by flow cytometry. The ES cell colonies were trypsinized and permeabilized prior to blocking and staining with the antibody (1ug/1.5 x 10 5 cells. A Cy3 conjugated anti-mouse antibody was used as the secondary.

    See Abreview

  • ICC/IF image of ab6002 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 (ab6002, 5µg/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) at a concentration of 1.43µM. This antibody also gave a positive result in 4% PFA fixed (10 min) Hek293, HepG2 and MCF7 cells at 5µg/ml, and in 100% methanol fixed (5 min) HeLa, Hek293, HepG2 and MCF7 cells at 5µg/ml.
  • Chromatin was prepared from nuclear lysate of the mouse embryonic stem cells. The cross-linking (X-ChiP) technique was used, crosslinking was performed for 15 minutes in 1% formaldehyde. The primary antibody was diluted to 0.0133µg/µg chromatin and incubated in ChIP Sonication Buffer with the sample for 24 hours at 4°C. The immunoprecipitated DNA was quantified by real time PCR.

    Cdx2: PCR primers situated in the promoter regions of Caudal type homeobox transcription factor 2
    Nef3: PCR primers situated in the promoter regions of neurofilament 3

    See Abreview

  • ICC/IF image of ab6002 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-Tween for 1h to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab6002, 5µg/ml) overnight at +4°C. The secondary antibody (green) was ab96879, DyLight® 488 goat anti-mouse 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.

References for Anti-Histone H3 (tri methyl K27) antibody [mAbcam 6002] - ChIP Grade (ab6002)

This product has been referenced in:
  • Chu CS  et al. O-GlcNAcylation regulates EZH2 protein stability and function. Proc Natl Acad Sci U S A 111:1355-60 (2014). Read more (PubMed: 24474760) »
  • Gonzalez ME  et al. EZH2 expands breast stem cells through activation of NOTCH1 signaling. Proc Natl Acad Sci U S A 111:3098-103 (2014). WB ; Human . Read more (PubMed: 24516139) »

See all 100 Publications for this product

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Application ChIP
Detection step Real-time PCR
Sample Rat Tissue lysate - whole (Gonad)
Specification Gonad
Negative control IgG
Type Cross-linking (X-ChIP)
Duration of cross-linking step: 0 second(s)
Positive control Input DNA
Username

Abcam user community

Verified customer

Submitted Jul 07 2014

Application ChIP on chip
Detection step Microarray
Sample Mouse Cell lysate - whole cell (Embryonic stem cells)
Specification Embryonic stem cells
Type Cross-linking (X-ChIP)
Duration of cross-linking step: 10 minute(s) and 0 second(s)
Specification of the cross-linking agent: formaldehyde
Positive control H3K4me3 and H3K9/14ac
Username

Mr. Dan Stummer

Verified customer

Submitted Apr 11 2014

Application ChIP
Detection step Real-time PCR
Sample Mouse Cell lysate - whole cell (Embryonic Stem Cells)
Specification Embryonic Stem Cells
Type Cross-linking (X-ChIP)
Duration of cross-linking step: 10 minute(s) and 0 second(s)
Specification of the cross-linking agent: formaldehyde
Username

Mr. Dan Stummer

Verified customer

Submitted Mar 28 2014

Application Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)
Blocking step Heat-inactivated normal donkey serum in 0.05% PBS-T as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 25°C
Antigen retrieval step None
Sample Mouse Tissue sections (Mouse, whole brain sections)
Specification Mouse, whole brain sections
Permeabilization Yes - Tween-20
Fixative Paraformaldehyde
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Submitted Mar 18 2014

Application Flow Cytometry
Fixation Paraformaldehyde
Permeabilization Yes - 0.1% Triton in PBS
Sample Human Cell (Primordial germ cells (PGCs))
Specification Primordial germ cells (PGCs)
Gating Strategy isotype control (black line in histogram)
Preparation Cell harvesting/tissue preparation method: accutase and accumax dissociation
Sample buffer: PBS 7.4 + 10% FBS for washing
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Submitted Mar 05 2014

Application Western blot
Loading amount 50 µg
Gel Running Conditions Reduced Denaturing (Bolt Gradient Gel)
Sample Human Cell lysate - whole cell (MDAMB231)
Specification MDAMB231
Blocking step BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 3% · Temperature: RT°C
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Submitted Dec 13 2013

Application Immunoprecipitation
Immuno-precipitation step Protein A/G
Sample Human Cell lysate - nuclear (MDAMB231)
Specification MDAMB231
Total protein in input 100 µg
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Submitted Dec 13 2013

Application Immunocytochemistry/ Immunofluorescence
Blocking step BSA as blocking agent for 30 minute(s) · Concentration: 2%
Sample Fruit fly (Drosophila melanogaster) Cell (Embrynic cells)
Specification Embrynic cells
Permeabilization Yes - 0.2% of Triton X100
Fixative Paraformaldehyde
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Submitted Oct 22 2013

Application Western blot
Loading amount 10 µg
Gel Running Conditions Non-reduced Non-Denaturing (Native) (18% gel)
Sample Human Cell lysate - other (Prostate cancer)
Specification Prostate cancer
Treatment 750uM GSK126
Blocking step Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 4°C
Username

Mr. Vineet Dhiman

Verified customer

Submitted Oct 07 2013

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