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Anti-Histone H3 (di methyl K4, tri methyl K4) antibody [mAbcam 6000] - ChIP Grade (ab6000)

Key features and details

  • Mouse monoclonal [mAbcam 6000] to Histone H3 (di methyl K4, tri methyl K4) - ChIP Grade
  • Suitable for: ICC/IF, WB, ChIP, Flow Cyt
  • Reacts with: Cow, Human, Rice
  • Isotype: IgG2b

Overview

  • Product name

    Anti-Histone H3 (di methyl K4, tri methyl K4) antibody [mAbcam 6000] - ChIP Grade
    See all Histone H3 primary antibodies

  • Description

    Mouse monoclonal [mAbcam 6000] to Histone H3 (di methyl K4, tri methyl K4) - ChIP Grade

  • Host species

    Mouse

  • Specificity

    This antibody detects a band of the appropriate size when used in Western blotting on a calf thymus histone preparation (see image). It is specifically blocked by peptides corresponding to di methylated K4 of Histone H3 and to tri methylated K4. Slight cross reactivity is observed with mono methyl K4 peptide in ELISA and Western Blot.

  • Tested Applications & Species

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

  • Immunogen

    Synthetic peptide within Human Histone H3 aa 1-100 (tri methyl K4) conjugated to keyhole limpet haemocyanin (Sulfosuccinimidyl 4-N-maleimidomethyl-cyclohexane-1-carboxylate (Sulfo-SMCC)). The exact sequence is proprietary. Hybridomas were prepared and the resulting clones were positively screened by ELISA against the immunising peptide and negatively screened against the non-modified equivalent peptide.
    (Peptide available as ab1342, ab7768)

  • Positive control

    • ChIP: Chromatin was prepared from U-2 OS cell . Flow Cyt: HeLa cells. WB: Calf thymus histone preparation nuclear lysate.

  • General notes

    This antibody clone is manufactured by Abcam.

    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

Images

  • ChIP - Anti-Histone H3 (di methyl K4, tri methyl K4) antibody [mAbcam 6000] - ChIP Grade (ab6000)
    ChIP - Anti-Histone H3 (di methyl K4, tri methyl K4) antibody [mAbcam 6000] - ChIP Grade (ab6000)

    Chromatin was prepared from U2OS cells according to the Abcam X-ChIP protocol. Cells were fixed with formaldehyde for 10min. The  ChIP was performed with 25 µg of chromatin, 2 µg of  ab6000 (blue), and 20 µl 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). Primers and probes are located in the first kb of the transcribed region.

  • Immunocytochemistry/ Immunofluorescence - Anti-Histone H3 (di methyl K4, tri methyl K4) antibody [mAbcam 6000] - ChIP Grade (ab6000)
    Immunocytochemistry/ Immunofluorescence - Anti-Histone H3 (di methyl K4, tri methyl K4) antibody [mAbcam 6000] - ChIP Grade (ab6000) This image is courtesy of Luke Hughes-Davies and Rhiannon Jade, Gurdon Institute, Cambridge, UK

    Immunofluorescent imaging of human cells (U2OS) with ab6000 reveals a diffuse background nuclear staining corresponding to global dimethylation of K4, with multiple foci of brighter staining.  The complete lack of nucleolar or cytoplasmic staining background confirms the high specificity of this 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.

    Due to the size constraints for images on our website, unfortunately we are unable to show a higher quality image.

  • Western blot - Anti-Histone H3 (di methyl K4, tri methyl K4) antibody [mAbcam 6000] - ChIP Grade (ab6000)
    Western blot - Anti-Histone H3 (di methyl K4, tri methyl K4) antibody [mAbcam 6000] - ChIP Grade (ab6000)
    All lanes : Anti-Histone H3 (di methyl K4, tri methyl K4) antibody [mAbcam 6000] - ChIP Grade (ab6000) 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 (ab7228) at 0.5 µg
    Lane 3 : Calf Thymus Histone Preparation Nuclear Lysate with Human Histone H3 (mono methyl K4) peptide (ab1340) at 0.5 µg
    Lane 4 : Calf Thymus Histone Preparation Nuclear Lysate with Human Histone H3 (di methyl K4) peptide (ab7768) at 0.5 µg
    Lane 5 : Calf Thymus Histone Preparation Nuclear Lysate with Human Histone H3 (tri methyl K4) peptide (ab1342) at 0.5 µg
    Lane 6 : Calf Thymus Histone Preparation Nuclear Lysate with Human Histone H3 (mono methyl K9) peptide (ab1771) at 0.5 µg
    Lane 7 : Calf Thymus Histone Preparation Nuclear Lysate with Human Histone H3 (di methyl K9) peptide (ab1772) at 0.5 µg
    Lane 8 : Calf Thymus Histone Preparation Nuclear Lysate with Human Histone H3 (tri methyl K9) peptide (ab1773) at 0.5 µg
    Lane 9 : Calf Thymus Histone Preparation Nuclear Lysate with Human Histone H3 (mono methyl K27) peptide (ab1780) at 0.5 µg
    Lane 10 : Calf Thymus Histone Preparation Nuclear Lysate with Human Histone H3 (di methyl K27) peptide (ab1781) at 0.5 µg
    Lane 11 : Calf Thymus Histone Preparation Nuclear Lysate with Human Histone H3 (tri methyl K27) peptide (ab1782) at 0.5 µg

    Lysates/proteins at 0.5 µg per lane.

    Secondary
    All lanes : Goat Anti-Mouse IgG H&L (HRP) preadsorbed (ab97040) at 1/10000 dilution

    Developed using the ECL technique.

    Performed under reducing conditions.

    Predicted band size: 15.2 kDa
    Observed band size: 17 kDa
    why is the actual band size different from the predicted?


    Exposure time: 20 minutes
  • Flow Cytometry - Anti-Histone H3 (di methyl K4, tri methyl K4) antibody [mAbcam 6000] - ChIP Grade (ab6000)
    Flow Cytometry - Anti-Histone H3 (di methyl K4, tri methyl K4) antibody [mAbcam 6000] - ChIP Grade (ab6000)

    Overlay histogram showing HeLa cells stained with ab6000 (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 (ab6000, 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 IgG2b [PLPV219] (ab91366, 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 (di methyl K4, tri methyl K4) antibody [mAbcam 6000] - ChIP Grade (ab6000)
    Immunocytochemistry/ Immunofluorescence - Anti-Histone H3 (di methyl K4, tri methyl K4) antibody [mAbcam 6000] - ChIP Grade (ab6000)

    Left image shows human Hep cell monolayer fixed with 4% formaldehyde, permeabilized using 0.5% Triton X-100, blocked with 5% FCS in PBS (20 min) and incubated with ab6000 for 1 hr (diluted 1/200 in PBS plus 1% FCS). Detection with anti-mouse rhodamin labeled secondary antibody. Unstained areas represent nucleoli and heterochromatin as judged from DAPI overlay of the same cell in the right image.

    This image was kindly supplied as part of the review submitted by Christian Schoefer.

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