H3K9me3 polyclonal antibody - Classic (sample size)

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10 µg
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Polyclonal antibody raised in rabbit against the region of histone H3 containing the trimethylated lysine 9 (H3K9me3), using a KLH-conjugated synthetic peptide.

Concentration1.52 µg/µl
Species reactivityHuman, mouse, zebrafish, Arabidopsis
PurityAffinity purified
PrecautionsThis product is for research use only. Not for use in diagnostic or therapeutic procedures.
Applications Suggested dilution References
ChIP * 1 μg/ChIP Fig 1, 2
ELISA 1:1,000 Fig 3
Dot Blotting 1:20,000 Fig 4
Western Blotting 1:1,000 Fig 5
Immunofluorescence 1:500 Fig 6
* Please note that the optimal antibody amount per IP should be determined by the end-user. We recommend testing 1-5 μg per IP.
  • Validation Data


    Figure 1. ChIP results obtained with the Diagenode antibody directed against H3K9me3
    ChIP assays were performed using human HeLa cells, the Diagenode antibody against H3K9me3 (Cat. No. C15410056) and optimized PCR primer sets for qPCR. ChIP was performed on sheared chromatin from 1 million HeLaS3 cells using the “Auto Histone ChIP-seq” kit (Cat. No. C01010022 ) on the SX-8G IP-Star automated system. A titration of the antibody consisting of 1, 2, 5, and 10 μg per ChIP experiment was analysed. IgG (2 μg/IP) was used as negative IP control. QPCR was performed with primers for the heterochromatin marker Sat2 and for the ZNF510 gene, used as positive controls, and for the promoters of the active c-fos and GAPDH genes, used as negative controls. Figure 1 shows the recovery, expressed as a % of input (the relative amount of immunoprecipitated DNA compared to input DNA after qPCR analysis).

    ChIP-seq figure A

    ChIP-seq figure B

    ChIP-seq figure C ChIP-seq figure D

    Figure 2. ChIP-seq results obtained with the Diagenode antibody directed against H3K9me3
    ChIP was performed with 0.5 μg of the Diagenode antibody against H3K9me3 (Cat. No. C15410056) on sheared chromatin from 1 million HeLa cells using the “iDeal ChIP-seq” kit (Cat. No. C01010051). The IP’d DNA was analysed by QPCR with optimized PCR primer pairs for the promoter regions of the active GAPDH and EIF4A2 genes, for the coding region of the ZNF510 gene and for the Sat2 satellite repeat (figure 2A). The IP’d DNA was subsequently analysed on an Illumina Genome Analyzer. Library preparation, cluster generation and sequencing were performed according to the manufacturer’s instructions. The 36 bp tags were aligned to the human genome using the BWA algorithm. Figure 2B shows the signal distribution along the long arm of chromosome 19 and a zoomin to an enriched region containing several ZNF repeat genes. Figure 2C and D show the enrichment at ZNF510 and ZNF12 on chromosome 9 and 7, respectively. These results clearly show an enrichment of H3K9me3 at ZNF repeat genes.


    Figure 3. Determination of the antibody titer
    To determine the titer of the antibody, an ELISA was performed using a serial dilution of the antibody directed against human H3K9me3 (Cat. No. C15410056), crude serum and flow through in antigen coated wells. The antigen used was a peptide containing the histone modification of interest. By plotting the absorbance against the antibody dilution (Figure 3), the titer of the antibody was estimated to be 1:60,000.

    Dot blot

    Figure 4. Cross reactivity tests using the Diagenode antibody directed against H3K9me3
    A Dot Blot analysis was performed to test the cross reactivity of the Diagenode antibody against H3K9me3 (Cat. No. C15410056) with peptides containing other modifications and unmodified sequences of histone H3 and H4. One hundred to 0.2 pmol of the peptide containing the respective histone modification were spotted on a membrane. The antibody was used at a dilution of 1:20,000. Figure 4 shows a high specificity of the antibody for the modification of interest.

    Western blot

    Figure 5. Western blot analysis using the Diagenode antibody directed against H3K9me3
    Western blot analysis was performed on histone extracts from HeLa cells (15 μg, lane 1) and on recombinant H3 (1 μg, lane2) using the Diagenode antibody against H3K9me3 (Cat. No. C15410056). The antibody was diluted 1:1,000 in TBS-Tween containing 5% skimmed milk. The position of the protein of interest is indicated on the right; the marker (in kDa) is shown on the left.


    Figure 6. Immunofluorescence using the Diagenode antibody directed against H3K9me3
    HeLa cells were stained with the Diagenode antibody against H3K9me3 (Cat. No. C15410056) and with DAPI. Cells were fixed with 4% formaldehyde for 10’ and blocked with PBS/TX-100 containing 5% normal goat serum and 1% BSA. The cells were immunofluorescently labelled with the H3K9me3 antibody (left) diluted 1:500 in blocking solution followed by an anti-rabbit antibody conjugated to Alexa488. The middle panel shows staining of the nuclei with DAPI. A merge of the two stainings is shown on the right.

  • Applications
    Enzyme-linked immunosorbent assay. Read more
    Dot blotting Read more
    Western blot : The quality of antibodies used in this technique is crucial for correct and specific protein identification. Diagenode offers huge selection of highly sensitive and specific western blot-validated antibodies. Learn more about: Load... Read more
    Immunofluorescence: Diagenode offers huge selection of highly sensitive antibodies validated in IF. Immunofluorescence using the Diagenode monoclonal antibody directed against CRISPR/Cas9 HeLa cells transfected with a Cas9 expression vector (... Read more
    ChIP-seq (ab)
    Read more
    ChIP-qPCR (ab)
    Read more
  • Documents
    Datasheet H3K9me3 C15410056 DATASHEET
    Polyclonal antibody raised in rabbit against the region of histone H3 containing the trimethylate...
    Antibodies you can trust POSTER
    Epigenetic research tools have evolved over time from endpoint PCR to qPCR to the analyses of lar...
    Epigenetic Antibodies Brochure BROCHURE
    More than in any other immuoprecipitation assays, quality antibodies are critical tools in many e...
  • Publications

    How to properly cite this product in your work

    Diagenode strongly recommends using this: H3K9me3 polyclonal antibody - Classic (sample size) (Diagenode Cat# C15410056-10 Lot# A1675-001P). Click here to copy to clipboard.

    Using our products in your publication? Let us know!

    Oestrogen receptor β regulates epigenetic patterns at specific genomic loci through interaction with thymine DNA glycosylase.
    Liu Y, Duong W, Krawczyk C, Bretschneider N, Borbély G, Varshney M, Zinser C, Schär P, Rüegg J.
    BACKGROUND: DNA methylation is one way to encode epigenetic information and plays a crucial role in regulating gene expression during embryonic development. DNA methylation marks are established by the DNA methyltransferases and, recently, a mechanism for active DNA demethylation has emerged involving the ten-ele...

    Epigenetic priming of inflammatory response genes by high glucose in adipose progenitor cells
    Rønningen T, Shah A, Reiner AH, Collas P, Moskaug JØ
    Cellular metabolism confers wide-spread epigenetic modifications required for regulation of transcriptional networks that determine cellular states. Mesenchymal stromal cells are responsive to metabolic cues including circulating glucose levels and modulate inflammatory responses. We show here that long term exposur...

    Polycomb RING1A- and RING1B-dependent histone H2A monoubiquitylation at pericentromeric regions promotes S-phase progression
    Bravo M, Nicolini F, Starowicz K, Barroso S, Calés C, Aguilera A, Vidal M
    The functions of polycomb products extend beyond their well-known activity as transcriptional regulators to include genome duplication processes. Polycomb activities during DNA replication and DNA damage repair are unclear, particularly without induced replicative stress. We have used a cellular model of conditional...

    Erosion of X Chromosome Inactivation in Human Pluripotent Cells Initiates with XACT Coating and Depends on a Specific Heterochromatin Landscape.
    Vallot C, Ouimette JF, Makhlouf M, Féraud O, Pontis J, Côme J, Martinat C, Bennaceur-Griscelli A, Lalande M, Rougeulle C
    Human pluripotent stem cells (hPSCs) display extensive epigenetic instability, particularly on the X chromosome. In this study, we show that, in hPSCs, the inactive X chromosome has a specific heterochromatin landscape that predisposes it to erosion of X chromosome inactivation (XCI), a process that occurs spontaneo...

    Embryonic stem cell differentiation requires full length Chd1.
    Piatti P, Lim CY, Nat R, Villunger A, Geley S, Shue YT, Soratroi C, Moser M, Lusser A
    The modulation of chromatin dynamics by ATP-dependent chromatin remodeling factors has been recognized as an important mechanism to regulate the balancing of self-renewal and pluripotency in embryonic stem cells (ESCs). Here we have studied the effects of a partial deletion of the gene encoding the chromatin remodel...

    The histone demethylase enzyme KDM3A is a key estrogen receptor regulator in breast cancer.
    Wade MA, Jones D, Wilson L, Stockley J, Coffey K, Robson CN, Gaughan L
    Endocrine therapy has successfully been used to treat estrogen receptor (ER)-positive breast cancer, but this invariably fails with cancers becoming refractory to treatment. Emerging evidence has suggested that fluctuations in ER co-regulatory protein expression may facilitate resistance to therapy and be involved i...

    TRIM28 Represses Transcription of Endogenous Retroviruses in Neural Progenitor Cells.
    Fasching L, Kapopoulou A, Sachdeva R, Petri R, Jönsson ME, Männe C, Turelli P, Jern P, Cammas F, Trono D, Jakobsson J
    TRIM28 is a corepressor that mediates transcriptional silencing by establishing local heterochromatin. Here, we show that deletion of TRIM28 in neural progenitor cells (NPCs) results in high-level expression of two groups of endogenous retroviruses (ERVs): IAP1 and MMERVK10C. We find that NPCs use TRIM28-mediated hi...

    Cryosectioning the intestinal crypt-villus axis: an ex vivo method to study the dynamics of epigenetic modifications from stem cells to differentiated cells
    Vincent A, Kazmierczak C, Duchêne B, Jonckheere N, Leteurtre E, Van Seuningen I
    The intestinal epithelium is a particularly attractive biological adult model to study epigenetic mechanisms driving adult stem cell renewal and cell differentiation. Since epigenetic modifications are dynamic, we have developed an original ex vivo approach to study the expression and epigenetic profiles of key gene...

    Analysis of an artificial zinc finger epigenetic modulator: widespread binding but limited regulation.
    Grimmer MR, Stolzenburg S, Ford E, Lister R, Blancafort P, Farnham PJ
    Artificial transcription factors (ATFs) and genomic nucleases based on a DNA binding platform consisting of multiple zinc finger domains are currently being developed for clinical applications. However, no genome-wide investigations into their binding specificity have been performed. We have created six-finger ATFs ...

    The PML-associated protein DEK regulates the balance of H3.3 loading on chromatin and is important for telomere integrity.
    Ivanauskiene K, Delbarre E, McGhie JD, Küntziger T, Wong LH, Collas P
    Histone variant H3.3 is deposited in chromatin at active sites, telomeres, and pericentric heterochromatin by distinct chaperones, but the mechanisms of regulation and coordination of chaperone-mediated H3.3 loading remain largely unknown. We show here that the chromatin-associated oncoprotein DEK regulates differen...

    The specific alteration of histone methylation profiles by DZNep during early zebrafish development.
    Ostrup O, Reiner AH, Aleström P, Collas P
    Early embryo development constitutes a unique opportunity to study acquisition of epigenetic marks, including histone methylation. This study investigates the in vivo function and specificity of 3-deazaneplanocin A (DZNep), a promising anti-cancer drug that targets polycomb complex genes. One- to two-cell stage embr...

    KMTase Set7/9 is a critical regulator of E2F1 activity upon genotoxic stress.
    Lezina L, Aksenova V, Ivanova T, Purmessur N, Antonov AV, Tentler D, Fedorova O, Garabadgiu AV, Talianidis I, Melino G, Barlev NA
    During the recent years lysine methyltransferase Set7/9 ((Su(var)-3-9, Enhancer-of-Zeste, Trithorax) domain containing protein 7/9) has emerged as an important regulator of different transcription factors. In this study, we report a novel function for Set7/9 as a critical co-activator of E2 promoter-binding factor 1...

    The histone variant composition of centromeres is controlled by the pericentric heterochromatin state during the cell cycle.
    Boyarchuk E, Filipescu D, Vassias I, Cantaloube S, Almouzni G
    Correct chromosome segregation requires a unique chromatin environment at centromeres and in their vicinity. Here, we address how the deposition of canonical H2A and H2A.Z histone variants is controlled at pericentric heterochromatin (PHC). Whereas in euchromatin newly synthesized H2A and H2A.Z are deposited through...

    RNAi-Mediated Gene silencing in Zebrafish Triggered by Convergent Transcription.
    Andrews OE, Cha DJ, Wei C, Patton JG
    RNAi based strategies to induce gene silencing are commonly employed in numerous model organisms but have not been extensively used in zebrafish. We found that introduction of transgenes containing convergent transcription units in zebrafish embryos induced stable transcriptional gene silencing (TGS) in cis and tran...

    Transcriptional Derepression of the ERVWE1 Locus following Influenza A Virus Infection.
    Li F, Nellåker C, Sabunciyan S, Yolken RH, Jones-Brando L, Johansson AS, Owe-Larsson B, Karlsson H
    UNLABELLED: Syncytin-1, a fusogenic protein encoded by a human endogenous retrovirus of the W family (HERV-W) element (ERVWE1), is expressed in the syncytiotrophoblast layer of the placenta. This locus is transcriptionally repressed in adult tissues through promoter CpG methylation and suppressive histone modificati...

    HDAC inhibitor confers radiosensitivity to prostate stem-like cells.
    Frame FM, Pellacani D, Collins AT, Simms MS, Mann VM, Jones G, Meuth M, Bristow RG, Maitland NJ
    Background:Radiotherapy can be an effective treatment for prostate cancer, but radiorecurrent tumours do develop. Considering prostate cancer heterogeneity, we hypothesised that primitive stem-like cells may constitute the radiation-resistant fraction.Methods:Primary cultures were derived from patients undergoing re...

    Lamin A/C-promoter interactions specify chromatin state-dependent transcription outcomes.
    Lund E, Oldenburg AR, Delbarre E, Freberg CT, Duband-Goulet I, Eskeland R, Buendia B, Collas P
    The nuclear lamina is implicated in the organization of the eukaryotic nucleus. Association of nuclear lamins with the genome occurs through large chromatin domains including mostly, but not exclusively, repressed genes. How lamin interactions with regulatory elements modulate gene expression in different cellular c...

    Long range epigenetic silencing is a trans-species mechanism that results in cancer specific deregulation by overriding the chromatin domains of normal cells.
    Forn M, Muñoz M, Tauriello DV, Merlos-Suárez A, Rodilla V, Bigas A, Batlle E, Jordà M, Peinado MA
    DNA methylation and chromatin remodeling are frequently implicated in the silencing of genes involved in carcinogenesis. Long Range Epigenetic Silencing (LRES) is a mechanism of gene inactivation that affects multiple contiguous CpG islands and has been described in different human cancer types. However, it is unkno...

    Transgene- and locus-dependent imprinting reveals allele-specific chromosome conformations.
    Lonfat N, Montavon T, Jebb D, Tschopp P, Nguyen Huynh TH, Zakany J, Duboule D
    When positioned into the integrin α-6 gene, an Hoxd9lacZ reporter transgene displayed parental imprinting in mouse embryos. While the expression from the paternal allele was comparable with patterns seen for the same transgene when present at the neighboring HoxD locus, almost no signal was scored at this integratio...

    Expression of a large LINE-1-driven antisense RNA is linked to epigenetic silencing of the metastasis suppressor gene TFPI-2 in cancer.
    Cruickshanks HA, Vafadar-Isfahani N, Dunican DS, Lee A, Sproul D, Lund JN, Meehan RR, Tufarelli C
    LINE-1 retrotransposons are abundant repetitive elements of viral origin, which in normal cells are kept quiescent through epigenetic mechanisms. Activation of LINE-1 occurs frequently in cancer and can enable LINE-1 mobilization but also has retrotransposition-independent consequences. We previously reported that i...

    Histone lysine trimethylation or acetylation can be modulated by phytoestrogen, estrogen or anti-HDAC in breast cancer cell lines.
    Dagdemir A, Durif J, Ngollo M, Bignon YJ, Bernard-Gallon D
    AIM: The isoflavones genistein, daidzein and equol (daidzein metabolite) have been reported to interact with epigenetic modifications, specifically hypermethylation of tumor suppressor genes. The objective of this study was to analyze and understand the mechanisms by which phytoestrogens act on chromatin in breast c...

    Epigenetic Regulation of Nestin Expression During Neurogenic Differentiation of Adipose Tissue Stem Cells.
    Boulland JL, Mastrangelopoulou M, Boquest AC, Jakobsen R, Noer A, Glover JC, Collas P.
    Adipose-tissue-derived stem cells (ASCs) have received considerable attention due to their easy access, expansion potential, and differentiation capacity. ASCs are believed to have the potential to differentiate into neurons. However, the mechanisms by which this may occur remain largely unknown. Here, we show that ...

    The histone demethylase Kdm3a is essential to progression through differentiation.
    Herzog M, Josseaux E, Dedeurwaerder S, Calonne E, Volkmar M, Fuks F
    Histone demethylation has important roles in regulating gene expression and forms part of the epigenetic memory system that regulates cell fate and identity by still poorly understood mechanisms. Here, we examined the role of histone demethylase Kdm3a during cell differentiation, showing that Kdm3a is essential for ...

    Global DNA hypomethylation coupled to repressive chromatin domain formation and gene silencing in breast cancer.
    Hon GC, Hawkins RD, Caballero OL, Lo C, Lister R, Pelizzola M, Valsesia A, Ye Z, Kuan S, Edsall LE, Camargo AA, Stevenson BJ, Ecker JR, Bafna V, Strausberg RL, Simpson AJ, Ren B
    While genetic mutation is a hallmark of cancer, many cancers also acquire epigenetic alterations during tumorigenesis including aberrant DNA hypermethylation of tumor suppressors, as well as changes in chromatin modifications as caused by genetic mutations of the chromatin-modifying machinery. However, the extent of...

    Prepatterning of developmental gene expression by modified histones before zygotic genome activation.
    Lindeman LC, Andersen IS, Reiner AH, Li N, Aanes H, Østrup O, Winata C, Mathavan S, Müller F, Aleström P, Collas P
    A hallmark of anamniote vertebrate development is a window of embryonic transcription-independent cell divisions before onset of zygotic genome activation (ZGA). Chromatin determinants of ZGA are unexplored; however, marking of developmental genes by modified histones in sperm suggests a predictive role of histone m...

    H3.5 is a novel hominid-specific histone H3 variant that is specifically expressed in the seminiferous tubules of human testes.
    Schenk R, Jenke A, Zilbauer M, Wirth S, Postberg J
    The incorporation of histone variants into chromatin plays an important role for the establishment of particular chromatin states. Six human histone H3 variants are known to date, not counting CenH3 variants: H3.1, H3.2, H3.3 and the testis-specific H3.1t as well as the recently described variants H3.X and H3.Y. We ...

    Embryonic lethal phenotype reveals a function of TDG in maintaining epigenetic stability.
    Cortázar D, Kunz C, Selfridge J, Lettieri T, Saito Y, MacDougall E, Wirz A, Schuermann D, Jacobs AL, Siegrist F, Steinacher R, Jiricny J, Bird A, Schär P
    Thymine DNA glycosylase (TDG) is a member of the uracil DNA glycosylase (UDG) superfamily of DNA repair enzymes. Owing to its ability to excise thymine when mispaired with guanine, it was proposed to act against the mutability of 5-methylcytosine (5-mC) deamination in mammalian DNA. However, TDG was also found to in...

    Characterization of the contradictory chromatin signatures at the 3' exons of zinc finger genes.
    Blahnik KR, Dou L, Echipare L, Iyengar S, O'Geen H, Sanchez E, Zhao Y, Marra MA, Hirst M, Costello JF, Korf I, Farnham PJ
    The H3K9me3 histone modification is often found at promoter regions, where it functions to repress transcription. However, we have previously shown that 3' exons of zinc finger genes (ZNFs) are marked by high levels of H3K9me3. We have now further investigated this unusual location for H3K9me3 in ZNF genes. Neither ...

    ZNF274 recruits the histone methyltransferase SETDB1 to the 3' ends of ZNF genes.
    Frietze S, O'Geen H, Blahnik KR, Jin VX, Farnham PJ
    Only a small percentage of human transcription factors (e.g. those associated with a specific differentiation program) are expressed in a given cell type. Thus, cell fate is mainly determined by cell type-specific silencing of transcription factors that drive different cellular lineages. Several histone modification...

    In vivo chromatin organization of mouse rod photoreceptors correlates with histone modifications.
    Kizilyaprak C, Spehner D, Devys D, Schultz P
    BACKGROUND: The folding of genetic information into chromatin plays important regulatory roles in many nuclear processes and particularly in gene transcription. Post translational histone modifications are associated with specific chromatin condensation states and with distinct transcriptional activities. The peculi...

    MicroChIP--a rapid micro chromatin immunoprecipitation assay for small cell samples and biopsies.
    Dahl JA, Collas P
    Chromatin immunoprecipitation (ChIP) is a powerful technique for studying protein-DNA interactions. Drawbacks of current ChIP assays however are a requirement for large cell numbers, which limits applicability of ChIP to rare cell samples, and/or lengthy procedures with limited applications. There are to date no pro...

    A rapid micro chromatin immunoprecipitation assay (microChIP).
    Dahl JA, Collas P
    Interactions of proteins with DNA mediate many critical nuclear functions. Chromatin immunoprecipitation (ChIP) is a robust technique for studying protein-DNA interactions. Current ChIP assays, however, either require large cell numbers, which prevent their application to rare cell samples or small-tissue biopsies, ...

    Fast genomic muChIP-chip from 1,000 cells
    Dahl JA, Reiner AH, Collas P.
    Genome-wide location analysis of histone modifications and transcription factor binding relies on chromatin immunoprecipitation (ChIP) assays. These assays are, however, time-consuming and require large numbers of cells, hindering their application to the analysis of many interesting cell types. We report here a fas...

    Promoter DNA Methylation Patterns of Differentiated Cells Are Largely Programmed at the Progenitor Stage
    Sørensen AL, Jacobsen BM, Reiner AH, Andersen IS, Collas P
    Mesenchymal stem cells (MSCs) isolated from various tissues share common phenotypic and functional properties. However, intrinsic molecular evidence supporting these observations has been lacking. Here, we unravel overlapping genome-wide promoter DNA methylation patterns between MSCs from adipose tissue, bone marrow...

    Chromatin Environment of Histone Variant H3.3 Revealed by Quantitative Imaging and Genome-scale Chromatin and DNA Immunoprecipitation
    Delbarre E, Jacobsen BM, Reiner AH, Sørensen AL, Kuntziger T, Collas P
    In contrast to canonical histones, histone variant H3.3 is incorporated into chromatin in a replication-independent manner. Posttranslational modifications of H3.3 have been identified; however, the epigenetic environment of incorporated H3.3 is unclear. We have investigated the genomic distribution of epitope-tagge...

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