H4K20me3 polyclonal antibody (sample size)

Catalog Number
10 µg
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Polyclonal antibody raised in rabbit against the region of histone H4 containing the trimethylated lysine 20 (H4K20me3), using a KLH-conjugated synthetic peptide.

Concentration1.07 µg/µl
Species reactivityHuman, mouse
PurityAffinity purified
PrecautionsThis product is for research use only. Not for use in diagnostic or therapeutic procedures.
Applications Suggested dilution References
ChIP/ChIP-seq * 1-2 μg/ChIP Fig 1, 2
CUT&TAG 1 μg Fig 3
ELISA 1:100 Fig 4
Dot Blotting 1:20,000 Fig 5
Western Blotting 1:1,000 Fig 6
Immunofluorescence 1:300 Fig 7

* 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

    H4K20me3 Antibody ChIP Grade

    Figure 1. ChIP results obtained with the Diagenode antibody directed against H4K20me3
    ChIP assays were performed using human HeLa cells, the Diagenode antibody against H4K20me3 (Cat. No. C15410057) and optimized PCR primer sets for qPCR. ChIP was performed with the “Auto Histone ChIP-seq” kit (Cat. No. C01010022) with sheared chromatin from 1 million cells using 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 (1 μg/IP) was used as negative IP control. QPCR was performed with primers for promoters of the active genes c-fos (Cat. No. C17011004) and GAPDH (Cat. No. C17011047), used as negative controls, and for the Sat2 satellite repeat region used as a positive control. Figure 1 shows the recovery, expressed as a % of input (the relative amount of immunoprecipitated DNA compared to input DNA after qPCR analysis).

    A.H4K20me3 Antibody for ChIP

    Figure 2. ChIP-seq results obtained with the Diagenode antibody directed against H4K20me3
    ChIP was performed with 1 μg of the Diagenode antibody against H4K20me3 (Cat. No. C15410057) on sheared chromatin from 1 million HeLaS3 cells using the “iDeal ChIP-seq” kit. The IP’d DNA was analysed by QPCR with optimized PCR primer pairs for the promoter and coding region of the active GAPDH gene, 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 ELAND 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 ZNF12 and ZNF510 on chromosome 7 and 9, respectively. These results clearly show an enrichment of H4K20me3 at ZNF repeat genes.

    B.H4K20me3 Antibody ChIP-seq Grade

    C.H4K20me3 Antibody for ChIP-seq

    D.H4K20me3 Antibody for ChIP-seq assay



    Figure 3. Cut&Tag results obtained with the Diagenode antibody directed against H4K20me3
    CUT&TAG (Kaya-Okur, H.S., Nat Commun 10, 1930, 2019) was performed on 50,000 K562 cells using 1 µg of the Diagenode antibody against H4K20me3 (cat. No. C15410057) and the Diagenode pA-Tn5 transposase (C01070001). The libraries were subsequently analysed on an Illumina NextSeq 500 sequencer (2x75 paired-end reads) according to the manufacturer's instructions. The tags were aligned to the human genome (hg19) using the BWA algorithm. Figure 3 shows the peak distribution on the long arm of chromosome 19 as well as a zoomin to a region enriched in ZNF repeat genes, and in a genomic region surrounding the MEG3 imprinted control gene on chromosome 14 (figure 3A and B, respectively).

    H4K20me3 Antibody ELISA validation

    Figure 4. Determination of the antibody titer
    To determine the titer of the antibody, an ELISA was performed using a serial dilution of the Diagenode antibody directed against H4K20me3 (Cat. No. C15410057), 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 4), the titer of the antibody was estimated to be 1:7,400.

    H4K20me3 Antibody validated in Dot Blot

    Figure 5. Cross reactivity test using the Diagenode antibody directed against H4K20me3
    A Dot Blot analysis was performed to test the cross reactivity of the Diagenode antibody against H4K20me3 (Cat. No. C15410057) with peptides containing other histone modifications and the unmodified H4K20. One hundred to 0.2 pmol of the respective peptides were spotted on a membrane. The antibody was used at a dilution of 1:20,000. Figure 5 shows a high specificity of the antibody for the modification of interest

    H4K20me3 Antibody validated for Western Blot

    Figure 6. Western blot analysis using the Diagenode antibody directed against H4K20me3
    Histone extracts of HeLa cells (15 μg) were analysed by Western blot using the Diagenode antibody against H4K20me3 (Cat. No. C15410057) 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.

    A.H4K20me3 Antibody validated in Immunofluorescence

    B.H4K20me3 Antibody validated in Immunofluorescence

    Figure 7. Immunofluorescence using the Diagenode antibody directed against H4K20me3
    Human osteosarcoma (U2OS) cells were stained with the Diagenode antibody against H4K20me3 (Cat. No. C15410057) and with DAPI. Cells were fixed with ice cold methanol for 10’ and blocked with PBS/TX-100 containing 5% normal goat serum. Figure 7A: cells were immunofluorescently labeled with the H4K20me3 antibody (left) diluted 1:300 in blocking solution followed by an anti-rabbit antibody conjugated to Alexa568 or with DAPI (right), which specifically labels DNA. Figure 6B: staining of the cells with the H4K20me3 antibody after incubation of the antibody with blocking peptide (Cat. No. C16000057), concentration: 5 ng/μl).

  • Target Description

    Histones are the main constituents of the protein part of chromosomes of eukaryotic cells. They are rich in the amino acids arginine and lysine and have been greatly conserved during evolution. Histones pack the DNA into tight masses of chromatin. Two core histones of each class H2A, H2B, H3 and H4 assemble and are wrapped by 146 base pairs of DNA to form one octameric nucleosome. Histone tails undergo numerous post-translational modifications, which either directly or indirectly alter chromatin structure to facilitate transcriptional activation or repression or other nuclear processes. In addition to the genetic code, combinations of the different histone modifications reveal the so-called “histone code”. Histone methylation and demethylation is dynamically regulated by respectively histone methyl transferases and histone demethylases.

  •  実験手法
    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
    CUT&Tagアッセイを成功させるための重要な要素の1つは使用される抗体の品質です。 特異性高い抗体は、目的のタンパク質のみをターゲットとした確実な結果を可能にします。 CUT&Tagで検証済みの抗体のセレクションはこちらからご覧ください。 Read more: Products for CUT&Tag assay Performance of Diagenode's antibodies in CUT&Tag Read more
  •  資料
    Datasheet H4K20me3 C15410057 DATASHEET
    Datasheet description
    Antibody ChIPSeq QC using the IPStar Compact POSTER
    Chromatin immunoprecipitation (ChIP) is the most widely used method to study protein-DNA interact...
    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...
  •  Safety sheets
    H4K20me3 antibody SDS US en Download
    H4K20me3 antibody SDS GB en Download
    H4K20me3 antibody SDS BE fr Download
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    H4K20me3 antibody SDS ES es Download
    H4K20me3 antibody SDS DE de Download
    H4K20me3 antibody SDS JP ja Download
    H4K20me3 antibody SDS BE nl Download
  •  出版物

    How to properly cite this product in your work

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    Histone H4K20 tri-methylation at late-firing origins ensures timely heterochromatin replication
    Brustel J. et al.
    Among other targets, the protein lysine methyltransferase PR-Set7 induces histone H4 lysine 20 monomethylation (H4K20me1), which is the substrate for further methylation by the Suv4-20h methyltransferase. Although these enzymes have been implicated in control of replication origins, the specific contribution of H4K2...

    Decoupling of DNA methylation and activity of intergenic LINE-1 promoters in colorectal cancer
    Vafadar-Isfahani N. et al.
    Hypomethylation of LINE-1 repeats in cancer has been proposed as the main mechanism behind their activation; this assumption, however, was based on findings from early studies that were biased toward young and transpositionally active elements. Here, we investigate the relationship between methylation of 2 intergeni...

    Heat shock represses rRNA synthesis by inactivation of TIF-IA and lncRNA-dependent changes in nucleosome positioning
    Zhao Z et al.
    Attenuation of ribosome biogenesis in suboptimal growth environments is crucial for cellular homeostasis and genetic integrity. Here, we show that shutdown of rRNA synthesis in response to elevated temperature is brought about by mechanisms that target both the RNA polymerase I (Pol I) transcription machinery and th...

    DOT1L Activity Promotes Proliferation and Protects Cortical Neural Stem Cells from Activation of ATF4-DDIT3-Mediated ER Stress In Vitro
    Roidl D, Hellbach N, Bovio PP, Villarreal A, Heidrich S, Nestel S, Grüning BA, Boenisch U, Vogel T
    Growing evidence suggests that the lysine methyltransferase DOT1L/KMT4 has important roles in proliferation, survival, and differentiation of stem cells in development and in disease. We investigated the function of DOT1L in neural stem cells (NSCs) of the cerebral cortex. The pharmacological inhibition and shRNA-me...

    Use of a mouse in vitro fertilization model to understand the developmental origins of health and disease hypothesis.
    Feuer SK, Liu X, Donjacour A, Lin W, Simbulan RK, Giritharan G, Piane LD, Kolahi K, Ameri K, Maltepe E, Rinaudo PF
    The Developmental Origins of Health and Disease hypothesis holds that alterations to homeostasis during critical periods of development can predispose individuals to adult-onset chronic diseases such as diabetes and metabolic syndrome. It remains controversial whether preimplantation embryo manipulation, clinically ...

    FSHD muscular dystrophy region gene 1 binds Suv4-20h1 histone methyltransferase and impairs myogenesis
    Neguembor M.V. et al.
    Facioscapulohumeral muscular dystrophy (FSHD) is an autosomal dominant myopathy with a strong epigenetic component. It is associated with deletion of a macrosatellite repeat leading to over-expression of the nearby genes. Among them, we focused on FSHD region gene 1 (FRG1) since its over-expression in mice, Xenopus ...

    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...

    Overexpression of facioscapulohumeral muscular dystrophy region gene 1 causes primary defects in myogenic stem cells.
    Xynos A, Neguembor MV, Caccia R, Licastro D, Nonis A, Di Serio C, Stupka E, Gabellini D
    Overexpression of facioscapulohumeral muscular dystrophy region gene 1 (FRG1) in mice, frogs and worms leads to muscular and vascular abnormalities. Nevertheless, the mechanism that follows FRG1 overexpression and finally leads to muscular defects is currently unknown. Here, we show that the earliest phenotype displ...

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