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

Key features and details

  • Mouse monoclonal [mAbcam 6147] to Histone H3 (di methyl K27, tri methyl K27)
  • Suitable for: ICC/IF, ELISA, WB, Flow Cyt
  • Reacts with: Mouse, Cow, Human
  • Isotype: IgG1

Overview

  • Product name

    Anti-Histone H3 (di methyl K27, tri methyl K27) antibody [mAbcam 6147]
    See all Histone H3 primary antibodies

  • Description

    Mouse monoclonal [mAbcam 6147] to Histone H3 (di methyl K27, tri methyl K27)

  • Host species

    Mouse

  • Specificity

    By Western blot, this antibody is blocked strongly by di and tri methyl K27 peptides and does not detect a band in Eed KO mouse ES cell lysates (which lack both di and tri methyl K27). All batches of ab6147 have >40% cross reactivity with both H3K27me2 and H3K27me3 as shown by ELISA. The sequence which it reacts with 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 Applications & Species

    Application Species
    Flow Cyt
    Human
    ICC/IF
    Human
    WB
    Cow
    Human
    See all applications and species data

  • Immunogen

    Synthetic peptide within Human Histone H3 aa 1-100 (tri methyl K27) conjugated to keyhole limpet haemocyanin (Sulfosuccinimidyl 4-N-maleimidomethyl-cyclohexane-1-carboxylate (Sulfo-SMCC)). The exact sequence is proprietary.
    (Peptide available as ab1782)

  • Positive control

    • Human Lung

  • General notes

    This antibody clone is manufactured by Abcam.

    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.

    If you require this antibody in a particular buffer formulation or a particular conjugate for your experiments, please contact orders@abcam.com or you can find further information here.

Properties

  • Form

    Liquid

  • Storage instructions

    Shipped 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 buffer

    pH: 7.40
    Preservative: 0.02% Sodium azide
    Constituent: PBS
  • Concentration information loading...

  • Purity

    IgG fraction

  • Clonality

    Monoclonal

  • Clone number

    mAbcam 6147

  • Isotype

    IgG1

  • Light chain type

    kappa

  • Research areas

Images

  • Western blot - Anti-Histone H3 (di methyl K27, tri methyl K27) antibody [mAbcam 6147] (ab6147)
    Western blot - Anti-Histone H3 (di methyl K27, tri methyl K27) antibody [mAbcam 6147] (ab6147)
    All lanes : Anti-Histone H3 (di methyl K27, tri methyl K27) antibody [mAbcam 6147] (ab6147) at 1 µg/ml

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

    Lysates/proteins at 0.5 µg per lane.

    Secondary
    All lanes : Goat polyclonal to Mouse IgG - H&L - Pre-Adsorbed (HRP) 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: 1 minute


    This blot was produced using a 4-12% Bis-tris gel under the MES buffer system. The gel was run at 200V for 35 minutes before being transferred onto a Nitrocellulose membrane at 30V for 70 minutes. The membrane was then blocked for an hour using 2% Bovine Serum Albumin before being incubated with ab6147 overnight at 4°C. Antibody binding was visualised using ECL development solution ab133406.
  • Immunocytochemistry/ Immunofluorescence - Anti-Histone H3 (di methyl K27, tri methyl K27) antibody [mAbcam 6147] (ab6147)
    Immunocytochemistry/ Immunofluorescence - Anti-Histone H3 (di methyl K27, 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).

  • Western blot - Anti-Histone H3 (di methyl K27, tri methyl K27) antibody [mAbcam 6147] (ab6147)
    Western blot - Anti-Histone H3 (di methyl K27, tri methyl K27) antibody [mAbcam 6147] (ab6147)
    All lanes : Anti-Histone H3 (di methyl K27, tri methyl K27) antibody [mAbcam 6147] (ab6147) at 1 µg/ml

    Lane 1 : HeLa (Human epithelial carcinoma cell line) Nuclear Lysate
    Lane 2 : HeLa (Human epithelial carcinoma cell line) Whole Cell Lysate
    Lane 3 : EED-/- mouse ES Whole Cell Lysate
    Lane 4 : WT mouse ES Whole Cell Lysate

    Lysates/proteins at 10 µg per lane.

    Secondary
    All lanes : Goat polyclonal to Mouse IgG - H&L - Pre-Adsorbed (HRP) 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: 10 seconds


    This blot was produced using a 4-12% Bis-tris gel under the MES buffer system. The gel was run at 200V for 35 minutes before being transferred onto a Nitrocellulose membrane at 30V for 70 minutes. The membrane was then blocked for an hour using 2% Bovine Serum Albumin before being incubated with ab6147 overnight at 4°C. Antibody binding was visualised using ECL development solution ab133406.

  • ELISA - Anti-Histone H3 (di methyl K27, tri methyl K27) antibody [mAbcam 6147] (ab6147)
    ELISA - Anti-Histone H3 (di methyl K27, tri methyl K27) antibody [mAbcam 6147] (ab6147)

    All batches of ab6147 are tested in ELISA against peptides to different Histone H3 modifications. Results show strong binding to Histone H3 - tri methyl K27 immunising peptide (ab1782) and Histone H3 - di methyl K27, indicating that this antibody specifically recognises both the Histone H3 - tri methyl K27 and di methyl K27 modifications. Binding is detected against the Histone H3 - di methyl K27 modification (>40%) (ab1781).

    ab2623 - Histone H3 - unmodified

    ab1340 - Histone H3 - mono methyl K4

    ab7768 - Histone H3 - di methyl K4

    ab1342 - Histone H3 - tri methyl K4

    ab1771 - Histone H3 - mono methyl K9

    ab1772 - Histone H3 - di methyl K9

    ab1773 - Histone H3 - tri methyl K9

    ab1780 - Histone H3 - mono methyl K27

    ab1781 - Histone H3 - di methyl K27

    ab1782 - Histone H3 - tri methyl K27

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

    See Abreview

  • Immunocytochemistry/ Immunofluorescence - Anti-Histone H3 (di methyl K27, tri methyl K27) antibody [mAbcam 6147] (ab6147)
    Immunocytochemistry/ Immunofluorescence - Anti-Histone H3 (di methyl K27, 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&degC. Samples were incubated with primary antibody (1/500 in diluent) for 14 hours at 4&degC. An FITC-conjugated donkey anti-mouse polyclonal IgG (1/300) was used as the secondary antibody.

    See Abreview

  • Flow Cytometry - Anti-Histone H3 (di methyl K27, tri methyl K27) antibody [mAbcam 6147] (ab6147)
    Flow Cytometry - Anti-Histone H3 (di methyl K27, 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 a goat anti-mouse DyLight® 488 (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.

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