Recombinant
RabMAb

Recombinant Anti-Histone H3 (tri methyl K4) antibody [EPR20551-225] - ChIP Grade (ab213224)

Overview

  • Product name

    Anti-Histone H3 (tri methyl K4) antibody [EPR20551-225] - ChIP Grade
    See all Histone H3 primary antibodies
  • Description

    Rabbit monoclonal [EPR20551-225] to Histone H3 (tri methyl K4) - ChIP Grade
  • Host species

    Rabbit
  • Tested applications

    Suitable for: ChIP, Flow Cyt, WB, ICC/IF, Dot blot, PepArr, IPmore details
  • Species reactivity

    Reacts with: Mouse, Rat, Human
  • Immunogen

    Synthetic peptide within Human Histone H3 aa 1-100 (tri methyl K4). The exact sequence is proprietary.
    Database link: P68431

  • Positive control

    • WB: HeLa, NIH/3T3 and C6 whole cell lysates. ICC/IF: HeLa and NIH/3T3 cells. Flow Cyt: HeLa cells. IP: NIH/3T3 whole cell lysate. ChIP: Chromatin prepared from HeLa cells.
  • General notes

    Our RabMAb® technology is a patented hybridoma-based technology for making rabbit monoclonal antibodies. For details on our patents, please refer to RabMab® patents.

    This product is a recombinant rabbit monoclonal antibody.

Properties

Applications

Our Abpromise guarantee covers the use of ab213224 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 µg for 25 µg of chromatin.
Flow Cyt 1/500.
WB 1/1000. Detects a band of approximately 15 kDa (predicted molecular weight: 15 kDa).

We recommend to use 2% BSA as blocking and antibody dilution buffer.

ICC/IF 1/500.
Dot blot 1/1000.
PepArr Use a concentration of 0.1 µg/ml.
IP 1/30.

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 Hela (human epithelial cel line from cervix adenocarcinoma) cells according to the Abcam X-ChIP protocol. Cells were fixed with formaldehyde for 10 min. The ChIP was performed with 25 μg of chromatin, 2 μg of ab213224(red), and 20 μl of Anti-rabbit IgG sepharose beads. 2 μg of Rabbit normal IgG was added to the beads control (grey). The immunoprecipitated DNA was quantified by real time PCR (Taqman approach). Primers and probes are located in the first kb of the transcribed region.

  • Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1%, Triton X-100-permeabilized NIH/3T3 (mouse embryo fibroblast cell line) cells labeling Histone H3 (tri methyl K4) with ab213224 at 1/500 dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) (ab150077) secondary antibody at 1/1000 dilution (green). Positive nuclear staining on NIH/3T3 cell lines.

    The nuclear counter stain is DAPI (blue). Tubulin is detected with Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor® 594) (ab195889) (red) at 1/200 dilution.

    Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) (ab150077) secondary antibody at 1/1000 dilution.

  • All lanes : Anti-Histone H3 (tri methyl K4) antibody [EPR20551-225] - ChIP Grade (ab213224) at 1/5000 dilution

    Lane 1 : HeLa (human epithelial cell line from cervix adenocarcinoma) whole cell lysate
    Lane 2 : NIH/3T3 (mouse embryo fibroblast cell line) whole cell lysate
    Lane 3 : C6 (rat glial tumor cell line) whole cell lysate

    Lysates/proteins at 10 µg per lane.

    Secondary
    All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution

    Developed using the ECL technique.

    Predicted band size: 15 kDa
    Observed band size: 15 kDa


    Exposure time: 1 second


    Blocking/Dilution buffer: 5% BSA/TBST.

  • All lanes : Anti-Histone H3 (tri methyl K4) antibody [EPR20551-225] - ChIP Grade (ab213224) at 1/5000 dilution

    Lane 1 : HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysates with 5% NFDM/TBST
    Lane 2 : HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysates with 2% BSA/TBST

    Lysates/proteins at 15 µg per lane.

    Secondary
    All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution (Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated)

    Predicted band size: 15 kDa


    Exposure time: 40 seconds


    We recommend to use 2% BSA as blocking and antibody dilution buffer.

  • ab213224 was tested in Peptide array against 501 different modified and unmodified histone peptides; each peptide is printed on the array at six concentrations (each in triplicate).

    Circle area represents affinity between the antibody and a peptide: all antigen-containing peptides are displayed as red circles, all other peptides as blue circles. The affinity is calculated as area under curve when antibody binding values are plotted against the corresponding peptide concentration. Each circle area is normalized to the peptide with the strongest affinity.

    The complete dataset, including full list of all peptides and information on the position of each peptide in the diagram, can be downloaded here.

  • Histone H3 (tri methyl K4) was immunoprecipitated from 0.35 mg of NIH/3T3 (mouse embryo fibroblast cell line) lysate with ab213224 at 1/30 dilution. Western blot was performed from the immunoprecipitate using ab213224 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/5000 dilution

    Lane 1: NIH/3T3 whole cell lysate 10 µg (Input). 

    Lane 2: ab213224 IP in NIH/3T3 whole cell lysate. 

    Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab213224 in NIH/3T3 whole cell lysate.

     

    Exposure time: 3 minutes.

    Blocking and dilution buffer and concentration: 5% BSA/TBST.

  • Dot blot analysis of Histone H3 (tri methyl K4) labeled with ab213224 at 1/1000 dilution.

    Lane 1: Histone H3K4Me3 peptide.

    Lane 2: Histone H3 unmodified peptide.

    Lane 3: Histone H3K(18+K36)Me3 peptide.

    Lane 4: Histone H3K18Me3 peptide.

    Lane 5: Histone H3K4Me2 peptide.

    Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution was used as secondary antibody.

     

    Exposure time: 3 minutes.

    Blocking and dilution buffer: 5% NFDM/TBST.

     

  • Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100-permeabilized HeLa (human epithelial cell line from cervix adenocarcinoma) cells labeling Histone H3 (tri methyl K4) with ab213224 at 1/500 dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) (ab150077) secondary antibody at 1/1000 dilution (green). Positive nuclear staining on HeLa cell line. 

    The nuclear counter stain is DAPI (blue). Tubulin is detected with Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor® 594) (ab195889) (red) at 1/200 dilution.

    Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) (ab150077) secondary antibody at 1/1000 dilution.

  • Flow cytometric analysis of 4% paraformaldehyde-fixed, 90% methanol-permeabilized HeLa (human epithelial cell line from cervix adenocarcinoma) cell line labeling Histone H3 (tri methyl K4) with ab213224 at 1/500 dilution (red) compared with a Rabbit IgG, monoclonal [EPR25A] - Isotype Control (ab172730) (black) and an unlabeled control (cells without incubation with primary antibody and secondary antibody) (blue). Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) (ab150077) at 1/2000 dilution was used as the secondary antibody.

References

This product has been referenced in:

  • Zhang XD  et al. The interaction of lncRNA EZR-AS1 with SMYD3 maintains overexpression of EZR in ESCC cells. Nucleic Acids Res 46:1793-1809 (2018). Read more (PubMed: 29253179) »
  • Yang L  et al. KDM6A and KDM6B play contrasting roles in nuclear transfer embryos revealed by MERVL reporter system. EMBO Rep 19:N/A (2018). Read more (PubMed: 30389724) »
See all 2 Publications for this product

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