Anti-Histone H3 (tri methyl K4) antibody [mAbcam12209] - ChIP Grade – BSA and Azide free (ab237971)

Overview

  • Product name

    Anti-Histone H3 (tri methyl K4) antibody [mAbcam12209] - ChIP Grade – BSA and Azide free
    See all Histone H3 primary antibodies
  • Description

    Mouse monoclonal [mAbcam12209] to Histone H3 (tri methyl K4) - ChIP Grade – BSA and Azide free
  • Host species

    Mouse
  • Specificity

    ab12209 is strongly blocked in Western blotting on histones by tri methyl K4, weakly by di methyl K4 and very weakly by mono methyl K4 peptides. It is not blocked by non-modified peptides. By ELISA the antibody binds to the tri methyl K4 peptide and at high antibody concentrations to di and mono methyl K4 peptides. It does not bind to unmodified, mono, di or tri methyl K9 or di or tri methyl K27 peptides. Not suitable for blocking with milk in Western blot (see Application notes).

  • Tested applications

    Suitable for: ChIP, WB, Flow Cyt, ICC/IF, ELISAmore details
  • Species reactivity

    Reacts with: Human
  • Immunogen

    Synthetic peptide within Histone H3 aa 1-100 (tri methyl K4) conjugated to keyhole limpet haemocyanin. The exact sequence is proprietary.

  • Positive control

    • FC: HeLa cells; ICC/IF: HeLa cells; WB: Calf Thymus Histone Preparation Nuclear Lysate; ChIP: U2OS cells.
  • General notes

    Ab237971 is a PBS only version of ab12209.

    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

  • Form

    Liquid
  • Storage instructions

    Shipped at 4°C. Store at +4°C short term (1-2 weeks). Store at +4°C. Do Not Freeze.
  • Storage buffer

    Constituent: PBS
  • Concentration information loading...
  • Purity

    IgG fraction
  • Clonality

    Monoclonal
  • Clone number

    mAbcam12209
  • Myeloma

    Sp2/0-Ag14
  • Isotype

    IgG1
  • Light chain type

    kappa

Applications

Our Abpromise guarantee covers the use of ab237971 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
ChIP Use 2-5 µg for 25 µg of chromatin.
WB Use a concentration of 1 - 5 µg/ml. Predicted molecular weight: 15 kDa.

NOT 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).

Flow Cyt Use 1µg for 106 cells.
ICC/IF Use a concentration of 5 µg/ml.
ELISA Use at an assay dependent concentration.

Target

  • Function

    Core 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 similarities

    Belongs to the histone H3 family.
  • Developmental stage

    Expressed 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 localization

    Nucleus. Chromosome.
  • Information by UniProt
  • Database links

  • Alternative names

    • H3 histone family member E pseudogene antibody
    • H3 histone family, member A antibody
    • H3/A antibody
    • H31_HUMAN antibody
    • H3F3 antibody
    • H3FA antibody
    • Hist1h3a antibody
    • HIST1H3B antibody
    • HIST1H3C antibody
    • HIST1H3D antibody
    • HIST1H3E antibody
    • HIST1H3F antibody
    • HIST1H3G antibody
    • HIST1H3H antibody
    • HIST1H3I antibody
    • HIST1H3J antibody
    • HIST3H3 antibody
    • histone 1, H3a antibody
    • Histone cluster 1, H3a antibody
    • Histone H3 3 pseudogene 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

Images

  • 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 ab12209 (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.

    This data was developed using the same antibody clone in a different buffer formulation containing PBS, L-arginine, and sodium azide (ab12209).

  • All lanes : Anti-Histone H3 (tri methyl K4) antibody [mAbcam12209] - ChIP Grade (ab12209) at 2 µg/ml

    Lane 1 : Calf Thymus Histone Preparation Nuclear Lysate
    Lane 2 : Calf Thymus Histone Preparation Nuclear Lysate with ab7228 at 0.25 µg/ml
    Lane 3 : Calf Thymus Histone Preparation Nuclear Lysate with ab1340 at 0.25 µg/ml
    Lane 4 : Calf Thymus Histone Preparation Nuclear Lysate with ab7768 at 0.25 µg/ml
    Lane 5 : Calf Thymus Histone Preparation Nuclear Lysate with ab1342 at 0.25 µg/ml
    Lane 6 : Calf Thymus Histone Preparation Nuclear Lysate with ab1771 at 0.25 µg/ml
    Lane 7 : Calf Thymus Histone Preparation Nuclear Lysate with ab1772 at 0.25 µg/ml
    Lane 8 : Calf Thymus Histone Preparation Nuclear Lysate with ab1773 at 0.25 µg/ml
    Lane 9 : Calf Thymus Histone Preparation Nuclear Lysate with ab1780 at 0.25 µg/ml
    Lane 10 : Calf Thymus Histone Preparation Nuclear Lysate with ab1781 at 0.25 µg/ml
    Lane 11 : Calf Thymus Histone Preparation Nuclear Lysate with ab1782 at 0.25 µg/ml

    Lysates/proteins at 0.5 µg per lane.

    Secondary
    All lanes : 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: 16 minutes


    This data was developed using the same antibody clone in a different buffer formulation containing PBS, L-arginine, and sodium azide (ab12209).

  • ICC/IF image of ab12209 stained human HeLa cells. The cells were 4% PFA fixed (10 min), permabilised in 0.1% PBS-Tween (20 min) and incubated with the antibody (ab12209, 5µg/ml) for 1h at room temperature. 1% BSA / 10% normal goat serum / 0.3M glycine was used to 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).

    This data was developed using the same antibody clone in a different buffer formulation containing PBS, L-arginine, and sodium azide (ab12209).

  • ELISA using ab12209 at varying antibody concentrations.

    Curve_SPL4  indicates binding to the tri methyl K4 peptide ab1342. In addition, SPL3 indicates partial binding to the di methyl K4 peptide ab7768.  There is very weak cross-reactivity with the mono methyl K4 peptide ab1340 (Curve_SPL2).

    Binding to the following peptides was not seen:
    SPL1 unmodified Histone H3, SPL5 Histone H3 mono methyl K9, SPL6 Histone H3 di methyl K9, SPL7 Histone H3 tri methyl K9, SPL8 Histone H3 mono methyl K27, SPL9 Histone H3 di methyl K27, SPL10 Histone H3 tri methyl K27.

    This data was developed using the same antibody clone in a different buffer formulation containing PBS, L-arginine, and sodium azide (ab12209).

  • Overlay histogram showing HeLa cells stained with ab12209 (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 (ab12209, 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 data was developed using the same antibody clone in a different buffer formulation containing PBS, L-arginine, and sodium azide (ab12209).

References

ab237971 has not yet been referenced specifically in any publications.

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