Anti-Histone H3 (di methyl K4) antibody - ChIP Grade (ab11946)

Overview

  • Product nameAnti-Histone H3 (di methyl K4) antibody - ChIP Grade
    See all Histone H3 primary antibodies
  • Description
    Goat polyclonal to Histone H3 (di methyl K4) - ChIP Grade
  • Tested applicationsIHC-P, ChIP, ICC/IF, WBmore details
  • Species reactivity
    Reacts with: Mouse, Rat, Cow, Human, Saccharomyces cerevisiae, Arabidopsis thaliana, Caenorhabditis elegans
    Predicted to work with: Chicken, Xenopus laevis, Fruit fly (Drosophila melanogaster), Schizosaccharomyces pombe, Zebrafish, a wide range of other species, Tobacco, Chlamydomonas reinhardtii, Aspergillus nidulans, Neurospora crassa
  • Immunogen

    Synthetic peptide conjugated to KLH derived from within residues 1 - 100 of Human Histone H3 di methylated at K4.

    (Peptide available as ab7768.)

  • Positive control
    • This antibody gave a positive signal in Calf Thymus Histone Preparation as well as the following whole cell lysates: PC12; NIH3T3; MEF1. It also gave a positive signal in FFPE human normal cervix tissue sections

Properties

Applications

Our Abpromise guarantee covers the use of ab11946 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
IHC-P Use a concentration of 1 µg/ml. Perform heat mediated antigen retrieval before commencing with IHC staining protocol.
ChIP Use 2-4 µg for 25 µg of chromatin.
ICC/IF 1/500. Use with paraformaldehyde or with methanol fixed cells.
WB Use a concentration of 1 µg/ml. Detects a band of approximately 17 kDa (predicted molecular weight: 15 kDa).Can be blocked with Histone H3 (di methyl K4) peptide (ab7768).

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 (di methyl K4) antibody - 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, 2µg of  ab11946 (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). Primers and probes are located in the first kb of the transcribed region.

  • All lanes : Anti-Histone H3 (di methyl K4) antibody - ChIP Grade (ab11946) at 1 µg/ml

    Lane 1 : NIH 3T3 (Mouse embryonic fibroblast cell line) Whole Cell Lysate
    Lane 2 : MEF1 (Mouse embryonic fibroblast cell line) Whole Cell Lysate

    Lysates/proteins at 15 µg per lane.

    Secondary
    Rabbit polyclonal to Goat IgG - H&L - Pre-Adsorbed (HRP) (ab65486) at 1/3000 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 : 2 minutes
  • Lane 1 : Marker
    Lanes 2 - 12 : Anti-Histone H3 (di methyl K4) antibody - ChIP Grade (ab11946) at 1 µg/ml

    Lane 1 : As above
    Lane 2 : Calf Thymus Histone Preparation Nuclear Lysate (ab121) at 0.5 µg
    Lane 3 : Calf Thymus Histone Preparation Nuclear Lysate (ab121) at 0.5 µg with Human Histone H3 (unmodified ) peptide (ab7228) at 0.75 µg/ml
    Lane 4 : Calf Thymus Histone Preparation Nuclear Lysate (ab121) at 0.5 µg with Human Histone H3 (mono methyl K4) peptide (ab1340) at 0.75 µg/ml
    Lane 5 : Calf Thymus Histone Preparation Nuclear Lysate (ab121) at 0.5 µg with Histone H3 (di methyl K4) peptide (ab7768) at 0.75 µg/ml
    Lane 6 : Calf Thymus Histone Preparation Nuclear Lysate (ab121) at 0.5 µg with Human Histone H3 (tri methyl K4) peptide (ab1342) at 0.75 µg/ml
    Lane 7 : Calf Thymus Histone Preparation Nuclear Lysate (ab121) at 0.5 µg with Human Histone H3 (mono methyl K9) peptide (ab1771) at 0.75 µg/ml
    Lane 8 : Calf Thymus Histone Preparation Nuclear Lysate (ab121) at 0.5 µg with Human Histone H3 (di methyl K9) peptide (ab1772) at 0.75 µg/ml
    Lane 9 : Calf Thymus Histone Preparation Nuclear Lysate (ab121) at 0.5 µg with Human Histone H3 (tri methyl K9) peptide (ab1773) at 0.75 µg/ml
    Lane 10 : Calf Thymus Histone Preparation Nuclear Lysate (ab121) at 0.5 µg with Histone H3 (mono methyl K27) peptide (ab1780) at 0.75 µg/ml
    Lane 11 : Calf Thymus Histone Preparation Nuclear Lysate (ab121) at 0.5 µg with Human Histone H3 (di methyl K27) peptide (ab1781) at 0.75 µg/ml
    Lane 12 : Calf Thymus Histone Preparation Nuclear Lysate (ab121) at 0.5 µg with Human Histone H3 (tri methyl K27) peptide (ab1782) at 0.75 µg/ml

    Secondary
    Lanes 2 - 12 : Rabbit polyclonal to Goat IgG (Alexa Fluor® 680) at 1/10000 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?)
  • Anti-Histone H3 (di methyl K4) antibody - ChIP Grade (ab11946) at 1 µg/ml + PC12 (Rat adrenal pheochromocytoma cell line) Whole Cell Lysate at 10 µg

    Secondary
    Goat Anti-Rabbit IgG H&L (HRP) preadsorbed (ab97080) at 1/5000 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 : 20 minutes
  • ab11946 (1/500) staining Histone H3 (di methyl K4) in assynchronous HeLa cells (green). Cells were fixed in paraformaldehyde, permeabilized with 0.5% Triton X100 and counterstained with DAPI in order to highlight the nucleus (red). For further experimental details please refer to Abreview.

    See Abreview

  • IHC image of Histone H3 staining in human normal cervix formalin fixed paraffin embedded tissue section, performed on a Leica BondTM system using the standard protocol B. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20 mins. The section was then incubated with ab11946, 1µg/ml, for 15 mins at room temperature. A donkey anti-goat biotinylated secondary antibody was used to detect the primary, and visualized using an HRP conjugated ABC system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX.

    For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.

References for Anti-Histone H3 (di methyl K4) antibody - ChIP Grade (ab11946)

This product has been referenced in:
  • Tie F  et al. Trithorax monomethylates histone H3K4 and interacts directly with CBP to promote H3K27 acetylation and antagonize Polycomb silencing. Development 141:1129-39 (2014). Read more (PubMed: 24550119) »
  • Ko YA  et al. Cytosine methylation changes in enhancer regions of core pro-fibrotic genes characterize kidney fibrosis development. Genome Biol 14:R108 (2013). ChIP ; Human . Read more (PubMed: 24098934) »

See all 7 Publications for this product

Product Wall

Abcam guarantees this product to work in the species/application used in this Abreview.
Application Immunocytochemistry/ Immunofluorescence
Sample Human Cell (HeLa)
Specification HeLa
Fixative Paraformaldehyde
Permeabilization Yes - 0.5% Triton X100
Username

Dr. Kirk McManus

Verified customer

Submitted Jun 09 2011

Abcam guarantees this product to work in the species/application used in this Abreview.
Application Western blot
Sample Saccharomyces cerevisiae Cell lysate - whole cell (1 OD of cells from liquid culture)
Loading amount 17 µg
Specification 1 OD of cells from liquid culture
Gel Running Conditions Reduced Denaturing (12%)
Blocking step Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: RT°C
Username

Dr. Marc Meneghini

Verified customer

Submitted Apr 20 2011

Abcam guarantees this product to work in the species/application used in this Abreview.
Application ChIP
Sample Human Cell lysate - whole cell (U2OS)
Specification U2OS
Type Cross-linking (X-ChIP)
Duration of cross-linking step: 10 minute(s) and 0 second(s)
Specification of the cross-linking agent: formaldehyde
Detection step Taqman
Positive control Histone H3 (ab1791)
Negative control beads
Username

Abcam user community

Verified customer

Submitted Jan 20 2009

Please note: All products are "FOR RESEARCH USE ONLY AND ARE NOT INTENDED FOR DIAGNOSTIC OR THERAPEUTIC USE"