Overview

  • Product name
    Anti-Histone H3 (di methyl K79) antibody - ChIP Grade
    See all Histone H3 primary antibodies
  • Description
    Rabbit polyclonal to Histone H3 (di methyl K79) - ChIP Grade
  • Host species
    Rabbit
  • Specificity
    ab3594 detects a 17 kDa band in single lane Western Blot. Peptide inhibition in Western Blot hasn't been processed. Modification specificity is determined by Peptide Array. ab3594 binds strongly to the Histone H3 dii methyl K79. In Peptide Array ab3594 also partially binds to mono methyl K79 and tri methyl K79 peptides.
  • Tested applications
    Suitable for: ChIP, ChIP/Chip, ICC/IF, WB, CHIPseq, IHC-P, PepArrmore details
  • Species reactivity
    Reacts with: Mouse, Cow, Human, Saccharomyces cerevisiae, Caenorhabditis elegans, Silk worm
    Predicted to work with: a wide range of other species, Mammals, Triticum aestivum
  • Immunogen

    Synthetic peptide within Human Histone H3 aa 50 to the C-terminus (di methyl K79) conjugated to Keyhole Limpet Haemocyanin (KLH). The exact sequence is proprietary.
    (Peptide available as ab4556)

  • Positive control
    • WB: Calf thymus histone preparation and HeLa whole cell extract. ICC/IF: blastocysts and HeLa cells ChIP: U2OS cells. IHC-P: Human breast carcinoma tissue.

Properties

  • Form
    Liquid
  • Storage instructions
    Shipped at 4°C. Store at +4°C short term (1-2 weeks). Upon delivery aliquot. Store at -20°C or -80°C. Avoid freeze / thaw cycle.
  • Storage buffer
    pH: 7.40
    Preservative: 0.02% Sodium azide
    Constituent: PBS

    Batches of this product that have a concentration < 1mg/ml may have BSA added as a stabilising agent. If you would like information about the formulation of a specific lot, please contact our scientific support team who will be happy to help.
  • Concentration information loading...
  • Purity
    Immunogen affinity purified
  • Clonality
    Polyclonal
  • Isotype
    IgG
  • Research areas

Applications

Our Abpromise guarantee covers the use of ab3594 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 at an assay dependent concentration.
ChIP/Chip Use at an assay dependent concentration.
ICC/IF Use a concentration of 1 µg/ml.
WB Use a concentration of 1 µg/ml. Detects a band of approximately 17 kDa (predicted molecular weight: 15 kDa).Can be blocked with Human Histone H3 (di methyl K79) peptide (ab4556).
CHIPseq Use at an assay dependent concentration. PubMed: 22196736
IHC-P Use a concentration of 1 µg/ml. Perform heat mediated antigen retrieval before commencing with IHC staining protocol.
PepArr Use a concentration of 0.2 - 0.02 µg/ml.

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

  • Verification of H3K79me2 antibody specificity.

    The commercial H3K79me2 primary antibody was preincubated with (+) or without (−) antigen peptide (Abcam, catalog no. ab4556, v/v = 5:1) at room temperature for 1.5 h before the incubation with IVF blastocysts. H3K79me2 signals were observed in blastocysts using unabsorbed primary antibody. By contrast, H3K79me2 signals were absent in blastocysts using pre-absorbed primary antibody. H3K79me2 antibody was localized with Alexa Flour 488-conjugated secondary antibody (green). DNA was stained with propidium iodide (red). Bottom panels showed the merged images (yellow) between H3K79me2 signals (green) and DNA staining (red). Scale bars = 50μm.

  • Chromatin was prepared from U2OS 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  ab3594 (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.

  • Anti-Histone H3 (di methyl K79) antibody - ChIP Grade (ab3594) at 20 µg + HeLa cell lysate at 20 µg

    Secondary
    HRP-conjugated Goat anti-rabbit IgG polyclonal at 1/2000 dilution

    Developed using the ECL technique.

    Performed under reducing conditions.

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


    Exposure time: 1 minute

    See Abreview

  • ICC/IF image of ab3594 stained HeLa cells. The cells were 100% methanol fixed (5 min) and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab3594, 1µg/ml) overnight at +4°C. 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) at a 1/200 dilution for 1h. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43µM. This antibody also gave a positive result in 100% methanol fixed (5 min) Hek293, HepG2 and MCF7 cells at 1µg/ml, and in 4% PFA fixed (10 min) HeLa, Hek293, HepG2 and MCF7 cells at 1µg/ml.
  • IHC image of Histone H3 (di 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 ab3594, 1µg/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.
  • All batches of ab3594 are tested in Peptide Array against peptides to different Histone H3 modifications. Six dilutions of each peptide are printed on to the Peptide Array in triplicate and results are averaged before being plotted on to a graph. Results show strong binding to Histone H3 - di methyl K79 peptide (ab4556), indicating that this antibody specifically recognises the Histone H3 - di methyl K79 modification.

    ab4556 - Histone H3 - di methyl K79

    ab4555 - Histone H3 - mono methyl K79

    ab4557 - Histone H3 - tri methyl K79

    ab4560 - Histone H4 - di methyl K79

    ab1772 - Histone H3 - di methyl K9

    ab4558 - Histone H3 - unmodified

  • Anti-Histone H3 (di methyl K79) antibody - ChIP Grade (ab3594) at 1 µg/ml + Calf Thymus Histone Preparation Nuclear Lysate (ab121) at 0.5 µg

    Secondary
    Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/10000 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: 30 seconds
  • ChIP analysis using ab3594 binding Histone H3 in HeLa cell nuclear lysate. Cells were cross-linked for 10 minutes with formaldehyde. Samples were incubated with primary antibody (5µg/µg chromatin) for 12 hours at 4°C. Protein binding was detected using real-time PCR.
    Positive control: 5'UTR of transcribed gene.
    Negative Control: Intergenic region.

    See Abreview

References

This product has been referenced in:
See all 143 Publications for this product

Customer reviews and Q&As

1-10 of 14 Abreviews or Q&A

Application
ELISA
Sample
Human Purified protein (PBCMs)
Specification
PBCMs
Type
Sandwich (Capture)
Blocking step
Milk as blocking agent for 1 hour(s) and 30 minute(s) · Concentration: 3%

Abcam user community

Verified customer

Submitted Aug 06 2014

Application
Western blot
Loading amount
20 µg
Gel Running Conditions
Reduced Denaturing (12)
Sample
Human Cell lysate - whole cell (Human Hela cells)
Specification
Human Hela cells
Blocking step
Milk as blocking agent for 12 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 4°C

Abcam user community

Verified customer

Submitted Oct 18 2013

Abcam guarantees this product to work in the species/application used in this Abreview.
Application
ChIP
Detection step
Real-time PCR
Sample
Human Cell lysate - nuclear (HeLa)
Specification
HeLa
Negative control
Intergenic region
Type
Cross-linking (X-ChIP)
Duration of cross-linking step: 10 minute(s) and 0 second(s)
Specification of the cross-linking agent: Formaldehyde
Positive control
5'UTR of a transcribed gene

Abcam user community

Verified customer

Submitted Jul 01 2013

Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Western blot
Sample
Caenorhabditis elegans Tissue lysate - nuclear (C. elegans)
Loading amount
10 µg
Specification
C. elegans
Treatment
0, 75Gy r-rays
Gel Running Conditions
Reduced Denaturing (12%)
Blocking step
Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 3% · Temperature: 25°C

Ms. Jinsun Ryu

Verified customer

Submitted Sep 12 2012

Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Western blot
Sample
Human Cell lysate - nuclear (Liver)
Loading amount
20 µg
Specification
Liver
Treatment
X protein was knockdowned
Gel Running Conditions
Reduced Denaturing (15)
Blocking step
Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: RT°C

Abcam user community

Verified customer

Submitted Feb 22 2011

Abcam guarantees this product to work in the species/application used in this Abreview.
Application
ChIP
Sample
Human Cell lysate - nuclear (K562)
Specification
K562
Type
Cross-linking (X-ChIP)
Duration of cross-linking step: 10 minute(s) and 0 second(s)
Specification of the cross-linking agent: 1% formaldehyde
Detection step
Other

Abcam user community

Verified customer

Submitted Nov 01 2010

Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Immunocytochemistry/ Immunofluorescence
Sample
Human Cell (293 cells)
Specification
293 cells
Fixative
Paraformaldehyde
Permeabilization
Yes - 1% SDS
Blocking step
BSA as blocking agent for 30 minute(s) · Concentration: 1% · Temperature: RT°C

Dr. Kathrin Bernt

Verified customer

Submitted Dec 11 2008

Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Immunocytochemistry/ Immunofluorescence
Sample
Mouse Cell (10T1/2)
Specification
10T1/2
Fixative
Paraformaldehyde
Permeabilization
Yes - 0.5% Triton X100 in PBS

Dr. Kirk Mcmanus

Verified customer

Submitted Sep 12 2008

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 in PBS

Dr. Kirk Mcmanus

Verified customer

Submitted Sep 12 2008

Abcam has not validated the combination of species/application used in this Abreview.
Application
Immunoprecipitation
Sample
Caenorhabditis elegans Cell lysate - whole cell (Embryo)
Total protein in input
250 µg
Specification
Embryo
Immuno-precipitation step
Protein A

Dr. Caroline Thivierge

Verified customer

Submitted Jan 25 2008

1-10 of 14 Abreviews or Q&A

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