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Anti-Histone H3 (methylated) antibody - ChIP Grade (ab28940)

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Overview

  • Product nameAnti-Histone H3 (methylated) antibody - ChIP GradeSee all Histone H3 (methylated) primary antibodies ...
  • Description
    Rabbit polyclonal to Histone H3 (methylated) - ChIP Grade
  • SpecificityReacts with tri-, di- and mono-methyl K79 of histone H3.
  • Tested applicationsWB, ChIP, ICC/IF, IHC-P more details
  • Species reactivity
    Reacts with: Cow
    Predicted to work with: Mouse, Rat, Rabbit, Chicken, Human, Pig, Saccharomyces cerevisiae, Caenorhabditis elegans, Fruit fly (Drosophila melanogaster), Schizosaccharomyces pombe, Shark, Rainbow Trout, Common Wheat (Triticum aestivum)
  • Immunogen

    Synthetic peptide conjugated to KLH derived from within residues 50 to the C-terminus of Human Histone H3.

    Read Abcam's proprietary immunogen policy

    (Peptide available as ab4557.)

  • Positive controlCalf Thymus Histone Preparation; Hela whole cell extract

Properties

Applications

Our Abpromise guarantee covers the use of ab28940 in the following tested applications.

The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.

Application Notes
WB
ChIP
ICC/IF
IHC-P
  • Application notesIHC-P: Use at a concentration of 5 µg/ml. Perform heat mediated antigen retrieval before commencing with IHC staining protocol.
    ChIP: Use 4-6 for 25µg for µg of chromatin
    ICC/IF: Use at a concentration of 1 µg/ml.
    WB: Use at a concentration of 1 µg/ml. Detects a band of approximately 15 kDa (predicted molecular weight: 15 kDa).


    Not yet tested in other applications.
    Optimal dilutions/concentrations should be determined by the end user.

    • 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.

      • Target information above from: UniProt accession P68431 The UniProt Consortium
      The Universal Protein Resource (UniProt) in 2010
      Nucleic Acids Res. 38:D142-D148 (2010) .

      Information by UniProt
    • Database links
      see all
    • Alternative names
        FLJ92264 antibodyH3 histone antibodyH3 histone antibody
        H3 histone family member E pseudogene antibodyH3 histone family member E pseudogene antibodyH3 histone family, member A antibodyH3/a antibodyH3/b antibodyH3/c antibodyH3/d antibodyh3/f antibodyH3/h antibodyH3/i antibodyH3/j antibodyH3/k antibodyH3/l antibodyH31_HUMAN antibodyH3F1K antibodyH3F3 antibodyH3FA antibodyH3FB antibodyH3FC antibodyH3FD antibodyH3FF antibodyH3FH antibodyH3FI antibodyH3FJ antibodyH3FK antibodyH3FL antibodyHIST1H3A antibodyHIST1H3B antibodyHIST1H3C antibodyHIST1H3D antibodyHIST1H3E antibodyHIST1H3F antibodyHIST1H3G antibodyHIST1H3H antibodyHIST1H3I antibodyHIST1H3J antibodyHIST3H3 antibodyHistone 1, H3a antibodyHistone cluster 1, H3a antibodyHistone cluster 1, H3b antibodyHistone cluster 1, H3c antibodyHistone cluster 1, H3d antibodyHistone cluster 1, H3e antibodyHistone cluster 1, H3f antibodyHistone cluster 1, H3g antibodyHistone cluster 1, H3i antibodyHistone cluster 1, H3j antibodyHistone H 3 antibodyHistone H3 3 pseudogene antibodyHistone H3 3 pseudogene antibodyHistone H3.1 antibodyHistone H3.1 antibodyHistone H3/a antibodyHistone H3/b antibodyHistone H3/c antibodyHistone H3/d antibodyHistone H3/f antibodyHistone H3/h antibodyHistone H3/i antibodyHistone H3/j antibodyHistone H3/k antibodyHistone H3/l antibody
      see all

    Anti-Histone H3 (methylated) antibody - ChIP Grade images

    • Western blot - Histone H3 (methyl K79) antibody (ab28940)
      Western blot - Histone H3 (methyl K79) antibody (ab28940)
      All lanes : Anti-Histone H3 (methylated) antibody - ChIP Grade (ab28940) at 1 µg/ml

      Lane 1 : Calf thymus histone lysate
      Lane 2 : Calf thymus histone lysate with Histone H3 peptide - mono methyl K79 (ab4555) at 1 µg/ml
      Lane 3 : Calf thymus histone lysate with Histone H3 peptide - di methyl K79 (ab4556) at 1 µg/ml
      Lane 4 : Calf thymus histone lysate with Histone H3 peptide - tri methyl K79 (ab4557) at 1 µg/ml
      Lane 5 : Calf thymus histone lysate with Histone H4 peptide - di methyl K79 (ab4560) at 1 µg/ml
      Lane 6 : Calf thymus histone lysate with Histone H3 peptide - tri methyl K4 (ab1342) at 1 µg/ml
      Lane 7 : Calf thymus histone lysate with Histone H3 peptide - unmodified (ab4558) at 1 µg/ml

      Lysates/proteins at 0.5 µg/ml per lane.

      Secondary
      Goat polyclonal Secondary Antibody to Rabbit IgG - H&L (HRP) (ab6721) at 1/5000 dilution

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


      Exposure time : 30 seconds
    • ChIP - Histone H3 (methyl K79) antibody - Limited Availability and ChIP Grade (ab28940)
      ChIP - Histone H3 (methyl K79) antibody - Limited Availability and ChIP Grade (ab28940)

      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  ab28940 (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.

    • Immunocytochemistry/ Immunofluorescence - Histone H3 (mono+di+tri methyl K79) antibody - Limited Availability and ChIP Grade (ab28940)
      Immunocytochemistry/ Immunofluorescence - Histone H3 (mono+di+tri methyl K79) antibody - Limited Availability and ChIP Grade (ab28940)

      ICC/IF image of ab28940 stained human HEK 293 cells. The cells were methanol fixed (5 min), permabilised in 0.1% PBS-Tween (20 min) and incubated with the antibody (ab28940, 1µg/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-rabbit 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 antibody also gave a positive IF result in HeLa, HepG2 and MCF7 cells.

    • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Histone H3 (mono+di+tri methyl K79) antibody - Limited Availability and ChIP Grade (ab28940)
      Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Histone H3 (mono+di+tri methyl K79) antibody - Limited Availability and ChIP Grade (ab28940)
      IHC image of Histone H3 (mono, di + tri methyl K79) staining in human breast carcinoma FFPE section, performed on a BondTM system using the standard protocol F. 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 ab28940, 5µg/ml, for 8 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX.

    References for Anti-Histone H3 (methylated) antibody - ChIP Grade (ab28940)

    This product has been referenced in:
    • Wirth M  et al. HIS-24 linker histone and SIR-2.1 deacetylase induce H3K27me3 in the Caenorhabditis elegans germ line. Mol Cell Biol 29:3700-9 (2009). IHC-Fr ; Caenorhabditis elegans . Read more (PubMed: 19380489) »

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