Anti-Histone H3 (tri methyl K9) antibody - ChIP Grade (ab8898)

Overview

  • Product nameAnti-Histone H3 (tri methyl K9) antibody - ChIP Grade
    See all Histone H3 primary antibodies
  • Description
    Rabbit polyclonal to Histone H3 (tri methyl K9) - ChIP Grade
  • SpecificitySpecific for Histone H3 tri methyl Lysine 9. Shows slight cross-reactivity with tri methyl K27, which shares a similar epitope (please see Western blot image). Does not react with mono or di methylated K9.
  • Tested applicationsIHC-Fr, IHC-P, ICC/IF, ChIP, WB, ChIP/Chip, Flow Cyt, CHIPseqmore details
  • Species reactivity
    Reacts with: Mouse, Rat, Human, Saccharomyces cerevisiae, Xenopus laevis, Fruit fly (Drosophila melanogaster), Indian Muntjac, Cyanidioschyzon merolae
    Predicted to work with: all Mammals
  • Immunogen

    Synthetic peptide conjugated to KLH derived from within residues 1 - 100 of Human Histone H3, tri methylated at K9.

    (Peptide available as ab1773.)

  • Positive control
    • Calf Thymus Histone Preparation; Hela whole cell extract IHC-P - Nornal human colon
  • General notesEvery new batch of this antibody is tested in house in ChIP.

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
    Note: 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
  • Clonality Polyclonal
  • IsotypeIgG
  • Research Areas

Applications

Our Abpromise guarantee covers the use of ab8898 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-Fr 1/50.
IHC-P 1/400. Perform heat mediated antigen retrieval before commencing with IHC staining protocol.
ICC/IF 1/500.
ChIP Use 2-4 µg for 25 µg of chromatin.
WB Use a concentration of 1 µg/ml. Detects a band of approximately 17 kDa (predicted molecular weight: 15.4 kDa).Can be blocked with Human Histone H3 (tri methyl K9) peptide (ab1773).
ChIP/Chip Use at an assay dependent concentration.
Flow Cyt 1/100.
CHIPseq Use at an assay dependent concentration. PubMed: 21795385

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 K9) antibody - ChIP Grade images

  • IHC image of ab8898 staining Histone H3 (tri methyl K9) 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 ab8898, 1/400 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

  • 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  ab8898 (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 for active and inactive loci, Sybr green approach for heterochromatic loci). Primers and probes are located in the first kb of the transcribed region.

  • All lanes : Anti-Histone H3 (tri methyl K9) antibody - ChIP Grade (ab8898) at 1 µg/ml

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

    Lysates/proteins at 0.5 µg per lane.

    Secondary
    IRDye 680 Conjugated Goat Anti-Rabbit IgG (H+L) at 1/10000 dilution

    Performed under reducing conditions.

    Predicted band size : 15.4 kDa
    Observed band size : 17 kDa (why is the actual band size different from the predicted?)
    Lane 8 shows that Rabbit polyclonal to Histone H3 (tri methyl K9) is blocked by the addition of the immunizing peptide (ab1773). Cross-reactivity with Histone H3 peptide - tri methyl K27 (ab1782) is also shown in Lane 11.
  • Indian muntjac fibroblast cells stained with anti-Histone H3 tri methyl K9, ab8898, (green, left panel, deconvolution image; red, right panel, epifluorescence image).

    The centromeres are enriched in Histone H3 tri methyl K9. There are also additional bands that occur throughout the chromosomes. Note that these images are taken in situ and are imaged under conditions where distinct cytogenetic-like banding patterns have not previously been possible to visualize (e.g., several acetylated antibodies have been reported to be associated with chromosome bands but, although not homogenously distributed along in situ chromosomes, they do not generate distinct banding patterns).
  • A 3-D reconstruction of a mouse embryonic fibroblast cell in metaphase stained with anti-Histone H3 tri methyl K9(green, ab8898) and DAPI (red).
  • ab8898 staining human uterine tumour tissue sections by IHC-P.  Sections were fixed in formaldehyde and subjected to heat mediated antigen retrieval in citrate buffer (pH 6) prior to blocking with 5% serum for 30 minutes at 22°C.  The primary antibody was diluted 1/400 and incubated with the sample for 30 minutes at 22°C.  A HRP-conjugated goat anti-rabbit antibody diluted 1/400, was used as the secondary.

    See Abreview

  • X-Chip assay was performed using nuclear lysates prepared from mouse ES cells. Crosslinking was done for 15 minutes in 1% formaldehyde. Primary antibody was incubated first with peptides ab7228, ab1342, ab1782, ab1773, ab1772 and ab1771 in a chip competition assay and then used in chip at 0.0133µg/ µg chromatin (chip sonication buffer) and incubated with sample for 24 hours at 4°C.

    Positive control: ChIP coupled with a peptide competition assay to validate the specificity of the antibody.

    Negative control: Genomic region (chr10:79154149-79155200) with no evidence of H3K9me3. 

    RT-PCR detection method was used.

    Polrmt: PCR primers situated in the coding regions of Polymerase (RNA) mitochondrial (DNA directed).

    Agrn: PCR primers situated in the coding regions of Agrin

  • ab8898 staining Histone H3 (tri methyl K9) in human differentiated haematopoietic stem cells by Flow Cytometry. Cells were fixed with paraformaldehyde and permeabilized with permeabilization buffer. The sample was incubated with the primary antibody (1/600) for 12 hours at 4°C. An Alexa Fluor® 488-conjugated goat polyclonal anti-rabbit IgG (1/1000) was used as the secondary antibody.
    Gating Strategy: Isotype negative control (white).

    See Abreview

  • These images were kindly submitted by Prof Bryan Turner, University of Birmingham. Undifferentiated Mouse Embryonic Stem cells or cells differentiated for 7 days were incubated with ab8898. The staining is specific for centromeric heterochromatin on metaphase chromosomes.
  • ICC/IF image of ab8898 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 (ab8898, 0.1µg/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) HepG2 and MCF7 cells at 0.1µg/ml and in 4% PFA fixed (10 min) HeLa, Hek293, HepG2 and MCF7 cells at 0.1ug/ml.
  • ICC/IF image of ab8898 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 (ab8898, 0.1µg/ml) overnight at +4°C. The secondary antibody (green) was ab96899, DyLight® 488 goat anti-rabbit IgG (H+L) used at a 1/250 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.

  • Anti-Histone H3 (tri methyl K9) antibody - ChIP Grade (ab8898) at 1/500 dilution + Rat E13 embryonic brain tissue lysate - nuclear at 10 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 : 15.4 kDa
    Observed band size : 15 kDa (why is the actual band size different from the predicted?)
    Additional bands at : 50 kDa (possible non-specific binding).

    Exposure time : 1 minute

    This image is courtesy of an anonymous Abreview

    See Abreview

  • All lanes : Anti-Histone H3 (tri methyl K9) antibody - ChIP Grade (ab8898) at 1/1000 dilution

    Lane 1 : Fruit fly embryo tissue lysate - nuclear at 4.5 µg
    Lane 2 : Fruit fly embryo tissue lysate - nuclear at 1.5 µg
    Lane 3 : Fruit fly embryo tissue lysate - nuclear at 0.5 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 : 15.4 kDa
    Observed band size : 15 kDa (why is the actual band size different from the predicted?)
    Additional bands at : 45 kDa (possible non-specific binding).

    Exposure time : 1 minute

    This image is courtesy of an anonymous Abreview

    See Abreview

References for Anti-Histone H3 (tri methyl K9) antibody - ChIP Grade (ab8898)

This product has been referenced in:
  • Baas R  et al. A novel microscopy-based high-throughput screening method to identify proteins that regulate global histone modification levels. J Biomol Screen 19:287-96 (2014). WB, ICC/IF ; Human . Read more (PubMed: 24334265) »
  • 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 275 Publications for this product

Product Wall

Application Western blot
Loading amount 20 µg
Gel Running Conditions Reduced Denaturing
Sample Rat Tissue lysate - nuclear (whole tissue extract)
Specification whole tissue extract
Blocking step Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 20°C
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Submitted Aug 05 2014

Application Western blot
Loading amount 20 µg
Gel Running Conditions Reduced Denaturing
Sample Fruit fly (Drosophila melanogaster) Tissue lysate - nuclear (whole tissue extract)
Specification whole tissue extract
Blocking step Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 20°C
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Submitted Aug 05 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
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Submitted Jul 07 2014

Application Immunocytochemistry/ Immunofluorescence
Blocking step Serum as blocking agent for 10 minute(s) · Concentration: 1% · Temperature: 21°C
Sample Human Cell (HEK293)
Specification HEK293
Permeabilization Yes - triton
Fixative Paraformaldehyde
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Submitted Apr 24 2014

Application Western blot
Loading amount 50 µg
Gel Running Conditions Reduced Denaturing (12 % GEL, SDS)
Sample Human Cell lysate - whole cell (HEK293)
Specification HEK293
Blocking step Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 21°C
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Submitted Apr 23 2014

Application Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)
Blocking step Heat-Inactivated Normal Donkey Serum as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 25°C
Antigen retrieval step None
Sample Mouse Tissue sections (Whole brain sections)
Specification Whole brain sections
Permeabilization Yes - Tween-20
Fixative Paraformaldehyde
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Submitted Apr 11 2014

Application ChIP
Detection step Real-time PCR
Sample Human Cell lysate - whole cell (SCC9 and OKF6)
Specification SCC9 and OKF6
Negative control IgG
Type Cross-linking (X-ChIP)
Duration of cross-linking step: 10 minute(s) and 0 second(s)
Specification of the cross-linking agent: formaldehyde
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Mr. Dan Stummer

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Submitted Apr 11 2014

Application Western blot
Loading amount 10 µg
Gel Running Conditions Reduced Denaturing
Sample Mouse Cell lysate - whole cell (Embryonic stem cells)
Specification Embryonic stem cells
Treatment vehicle control and all-trans retinoic acid (1uM, 24hrs)
Blocking step Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 21°C
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Mr. Dan Stummer

Verified customer

Submitted Apr 03 2014

Application ChIP
Detection step Real-time PCR
Sample Mouse Cell lysate - whole cell (F9 teratocarcinoma)
Specification F9 teratocarcinoma
Negative control IgG (set to 1.00)
Type Cross-linking (X-ChIP)
Duration of cross-linking step: 10 minute(s) and 0 second(s)
Specification of the cross-linking agent: paraformaldehyde
Positive control H3K4me3
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Mr. Dan Stummer

Verified customer

Submitted Mar 28 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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