Anti-Histone H3 (tri methyl K4) antibody - ChIP Grade (ab8580)

Overview

  • Product nameAnti-Histone H3 (tri methyl K4) antibody - ChIP GradeSee all Histone H3 primary antibodies ...
  • Description
    Rabbit polyclonal to Histone H3 (tri methyl K4) - ChIP Grade
  • Tested applicationsChIP/Chip, PepArr, ICC, ChIP, WB, IHC-Fr, IP, CHIPseq, Flow Cyt, ICC/IF, IHC-Pmore details
  • Species reactivity
    Reacts with: Mouse, Rat, Rabbit, Human, Pig, Saccharomyces cerevisiae, Tetrahymena sp., Xenopus laevis, Arabidopsis thaliana, Caenorhabditis elegans, Fruit fly (Drosophila melanogaster), Zebrafish, Trypanosoma cruzi, Marmoset (common), Rice
    Predicted to work with: Cow, Indian Muntjac, Oikopleura - a pelagic tunicate, Plants, all Mammals
  • Immunogen

    Synthetic peptide corresponding to Human Histone H3 aa 1-100 (tri methyl K4) conjugated to Keyhole Limpet Haemocyanin (KLH).
    (Peptide available as ab92374)

  • Positive control
    • Calf Thymus Histone Preparation ICC/IF - HeLa cells. IHC-P - Normal human colon
  • General notes

    In immunofluorescence, a distinct property of tri methyl lysine 4 is its apparent 'ringing' of regions that appear as nucleoplasmic 'holes'. These represent the positions of splicing factor compartments, which often are easy to identify using only DNA stains in Indian muntjac fibroblasts. These splicing factor compartments are known to be preferentially associated with active genes and highly acetylated histone H3. The antibody, as expected, fails to stain heterochromatin (work by Kirk McManus, lab of Michael Hendzel). The immunofluorescence results suggest this antibody is an exceptional euchromatin probe.

Properties

  • FormLiquid
  • Storage instructionsShipped 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 bufferpH: 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...
  • PurityImmunogen affinity purified
  • Primary antibody notes In immunofluorescence, a distinct property of tri methyl lysine 4 is its apparent 'ringing' of regions that appear as nucleoplasmic 'holes'. These represent the positions of splicing factor compartments, which often are easy to identify using only DNA stains in Indian muntjac fibroblasts. These splicing factor compartments are known to be preferentially associated with active genes and highly acetylated histone H3. The antibody, as expected, fails to stain heterochromatin (work by Kirk McManus, lab of Michael Hendzel). The immunofluorescence results suggest this antibody is an exceptional euchromatin probe.
  • Clonality Polyclonal
  • IsotypeIgG
  • Research Areas

Applications

Our Abpromise guarantee covers the use of ab8580 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/Chip Use at an assay dependent concentration.
PepArr Use a concentration of 0.2 - 2 µg/ml.
ICC Use at an assay dependent concentration.
ChIP Use 2 µg for 25 µg of chromatin.
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 (tri methyl K4) peptide (ab92374).
IF Use a concentration of 1 µg/ml.
IHC-Fr 1/500.
IP Use at an assay dependent concentration.
CHIPseq Use at an assay dependent concentration. PubMed: 20952408
Flow Cyt Use at an assay dependent concentration.
ICC/IF 1/100 - 1/5000.
IHC-P Use at an assay dependent concentration. PubMed: 17634443

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 (tri methyl K4) antibody - ChIP Grade images

  • IHC image of ab8580 staining Histone H3 (tri methyl K4) in normal human colon formalin-fixed paraffin-embedded tissue sections*, performed on a Leica Bond. 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 ab8580, 1/500 dilution, for 15 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. No primary antibody was used in the negative control (shown on the inset).

    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.

    *Tissue obtained from the Human Research Tissue Bank, supported by the NIHR Cambridge Biomedical Research Centre

  • ab8580 staining Histone H3 (tri methyl K4) in HeLa cells. All cells were fixed with 100% methanol (5min) and then blocked in 1% BSA/10% normal goat serum/0.3M glycine in 0.1%PBS-Tween for 1h. The cells were then incubated with ab4729 at 1/1000 and ab7291 at 1µg/ml overnight at +4°C, followed by a further incubation at room temperature for 1h with an AlexaFluor®488 Goat anti-Rabbit secondary (ab150077) at 2 μg/ml (shown in green) and AlexaFluor®594 Goat anti-Mouse secondary (ab150120) at 2 μg/ml (shown in pseudo color red). Nuclear DNA was labelled in blue with DAPI.

    Negative controls: 1– Rabbit primary and anti-mouse secondary antibody; 2 – Mouse primary antibody and anti-rabbit secondary antibody. Controls 1 and 2 indicate that there is no unspecific reaction between primary and secondary antibodies used.

  • 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  ab8580 (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.

  • All batches of ab8580 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 - tri methyl K4 peptide (ab1342), indicating that this antibody specifically recognises the Histone H3 - tri methyl K4 modification.

    1. ab1340 - Histone H3 - mono methyl K4
    2. ab1342 - Histone H3 - tri methyl K4
    3. ab1771 - Histone H3 - mono methyl K9
    4. ab1772 - Histone H3 - di methyl K9
    5. ab1773 - Histone H3 - tri methyl K9
    6. ab1780 - Histone H3 - mono methyl K27
    7. ab1781 - Histone H3 - di methyl K27
    8. ab1782 - Histone H3 - tri methyl K27
    9. ab7228 - Histone H3 - unmodified
    10. ab7768 - Histone H3 - di methyl K4
  • Anti-Histone H3 (tri methyl K4) antibody - ChIP Grade (ab8580) at 1 µg/ml + Calf Thymus Histone Preparation Nuclear Lysate at 0.5 µg

    Secondary
    Goat polyclonal to Rabbit 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 : 8 minutes
  • All lanes : Anti-Histone H3 (tri methyl K4) antibody - ChIP Grade (ab8580) at 1/5000 dilution

    Lane 1 : S. cerevisiae whole cell lysate with WT
    Lane 2 : S. cerevisiae whole cell lysate with set1
    Lane 3 : S. cerevisiae whole cell lysate with bre2
    Lane 4 : S. cerevisiae whole cell lysate with sdc1
    Lane 5 : S. cerevisiae whole cell lysate with shg1
    Lane 6 : S. cerevisiae whole cell lysate with spp1
    Lane 7 : S. cerevisiae whole cell lysate with swd1
    Lane 8 : S. cerevisiae whole cell lysate with swd3

    Lysates/proteins at 40 µg per lane.


    Performed under reducing conditions.

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

    John E. Mueller and J. Ruth German (Mary Bryk lab)

    Rabbit polyclonal to Histone H3 tri methyl K9 (ab8580) at 1/5000 on S. cerevisiae whole cell lysate (40 ug per lane).

    Protein resolved on 15% SDS-PAGE gel. After transfer to PVDF membrane, blots were blocked in 1X PBS, 0.1% Tween-20, and 5% milk. ab1791 was diluted in 5 ml blocking buffer at 1/5000.  Blots plus primary antibodies were either incubated overnight at 4C or at RT for 2 hr. Blots were washed 6X for 10 min each in PBS with 0.1% Tween-20 before addition of secondary antibodies. Secondary antibodies were diluted 1/2,000 in blocking buffer and incubated with blots for 2 hr at RT. Secondary blots were washed 4X for 10 min each in PBS with 0.1% Tween-20 and 2X for 10 min each in PBS.

    Protein resolved on 15% SDS-PAGE gel. After transfer to PVDF membrane, blots were blocked in 1X PBS, 0.1% Tween-20, and 5% milk. ab1791 was diluted in 5 ml blocking buffer at 1/5000. Blots plus primary antibodies were either incubated overnight at 4C or at RT for 2 hr.
  • ab8580 staining cultured human primary fibroblasts by ICC.  Cells were PFA fixed and permeabilized in TritonX100 and saponin prior to blocking with 1% BSA for 1 hour at RT.  The primary antibody was diluted 1/100 and incubated with the sample for 16 hours at 4°C.  A FITC-conjugated rabbit anti-rabbit IgG antibody was used as the secondary.

    See Abreview

  • Staining (green) with the anti-trimethyl Lysine K4 of Histone H3 antibody (ab8580) shows ringing of regions that appear as nucleoplasmic holes. These represent the positions of splicing factor compartments, which are preferentially associated with active genes and highly acetylated histone H3.   

    The antibody, as expected, fails to stain heterochromatin (red). 

  • IF of primary cultures of bladder cancer which arise from urothelial cells to determine whether the alterations in chromatin during cancer development would be enhanced by the histone modification antibodies. Both cell types gave very weak staining with the trimethyl-K9-H3 antibody.
  • Mouse zygotes stained with the Tri-Methyl K4 histone H3 antibody
    (green) and DNA (blue). This modification is readily detected in the two
    pronuclei of the zygote.

    This image was kindly supplied as part of the review submitted by Dr Maria Elena Torres Padilla (University of Cambridge, UK).

  • Human female lymphoblast immunostained with ab8580 (1:100)(yellowish green) specific for histone H3 lysine 4 (H3-K4) trimethylation; the DNA is stained red with propidium iodide (PI).Note the inactive X chromosome (arrow) and pericentromeric heterochromatin are largely devoid of this modification.
  • IHC-P image of Histone H3 (tri methyl K4) staining with ab8580 on tissue sections from adult marmoset testis. The sections were subjected to heat-mediated antigen retrieval using Dako antigen retrieval solution. The sections were blocked with 5% milk for 30 minutes at 25°C and then incubated with ab8580 (1/100 dilution) for 16 hours at 4°C. The secondary used was an Alexa-Fluor 555 conjugated goat anti-rabbit polyclonal.

    See Abreview

References for Anti-Histone H3 (tri methyl K4) antibody - ChIP Grade (ab8580)

This product has been referenced in:
  • Liu Y  et al. CHD7 interacts with BMP R-SMADs to epigenetically regulate cardiogenesis in mice. Hum Mol Genet 23:2145-56 (2014). ChIP ; Mouse . Read more (PubMed: 24293546) »
  • Harding JL  et al. Small RNA profiling of Xenopus embryos reveals novel miRNAs and a new class of small RNAs derived from intronic transposable elements. Genome Res 24:96-106 (2014). ChIP ; Xenopus laevis, Xenopus tropicalis . Read more (PubMed: 24065776) »

See all 359 Publications for this product

Product Wall

You are able to specify particular lots with your order which we are happy to supply provided these are in stock. You may request this by phone or if ordering online or by fax by adding the specific lot requested to the notes section of the order.

Application Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)
Sample Marmoset (common) Tissue sections (Adult marmoset testis)
Specification Adult marmoset testis
Fixative Formaldehyde
Antigen retrieval step Heat mediated - Buffer/Enzyme Used: Dako Antigen retrieval solution
Permeabilization No
Blocking step Milk as blocking agent for 30 minute(s) · Concentration: 5% · Temperature: 25°C
Username

Zachary Yu-Ching Lin

Verified customer

Submitted Aug 28 2012

Application Immunocytochemistry/ Immunofluorescence
Sample Human Cell (MCF-7)
Specification MCF-7
Fixative Formaldehyde
Permeabilization Yes - Triton-X-100
Blocking step Serum as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 1% · Temperature: 25°C
Username

Abcam user community

Verified customer

Submitted May 16 2012

Application Immunocytochemistry/ Immunofluorescence
Sample Mouse Cell (mouse ES cells)
Specification mouse ES cells
Fixative Methanol
Permeabilization No
Blocking step (agent) for 1 hour(s) and 0 minute(s) · Concentration: 0.2% · Temperature: 25°C
Username

Abcam user community

Verified customer

Submitted Mar 23 2012

Application Immunocytochemistry
Sample Human Cultured Cells (HeLa cells, human cervical carcinoma cells)
Specification HeLa cells, human cervical carcinoma cells
Fixative Formaldehyde
Permeabilization Yes - Triton
Blocking step Serum as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 2% · Temperature: 22°C
Username

Dr. Roman Hudec

Verified customer

Submitted Feb 09 2011

Application Western blot
Loading amount 10 µg
Gel Running Conditions Reduced Denaturing
Sample Human Cell lysate - nuclear (HeLa whole cell extract)
Specification HeLa whole cell extract
Blocking step BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 20°C
Username

Abcam user community

Verified customer

Submitted Jul 14 2014

Application ChIP
Detection step Real-time PCR
Sample Rat Tissue lysate - whole (Gonad)
Specification Gonad
Negative control IgG
Type Cross-linking (X-ChIP)
Duration of cross-linking step: 0 second(s)
Positive control Input DNA
Username

Abcam user community

Verified customer

Submitted Jul 07 2014

Application Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)
Blocking step Serum as blocking agent for 45 minute(s) · Concentration: 1.5µg/mL · Temperature: 25°C
Antigen retrieval step Enzymatic
Sample Rat Tissue sections (Gonad)
Specification Gonad
Permeabilization No
Fixative Formaldehyde
Username

Abcam user community

Verified customer

Submitted Apr 01 2014

Application Immunocytochemistry
Blocking step Serum as blocking agent for 20 minute(s) · Concentration: 5% · Temperature: 23°C
Sample Mouse Cultured Cells (Differentiating mouse embryonic fibroblasts)
Specification Differentiating mouse embryonic fibroblasts
Permeabilization Yes - 0.1% Triton X-100
Fixative Paraformaldehyde
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Verified customer

Submitted Feb 21 2014

Application Flow Cytometry
Fixation Paraformaldehyde
Permeabilization Yes - eBioscience Permeabilization Buffer
Sample Human Cell (Differentiated Haematopoietic Stem Cells)
Specification Differentiated Haematopoietic Stem Cells
Gating Strategy Isotype negative control (white)
Preparation Cell harvesting/tissue preparation method: Typsin-EDTA Cell Dissociation
Sample buffer: PBS and 10% FBS
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Submitted Feb 20 2014

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