H4K20me3 polyclonal antibody - Classic

Catalog Number
50 μg/47 μl
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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 * 1-2 μg/ChIP Fig 1, 2
ELISA 1:100 Fig 3
Dot Blotting 1:20,000 Fig 4
Western Blotting 1:1,000 Fig 5
Immunofluorescence 1:300 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 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).

    ChIP-seq figure A

    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.

    ChIP-seq figure B

    ChIP-seq figure C

    ChIP-seq figure D


    Figure 3. 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 3), the titer of the antibody was estimated to be 1:7,400.

    Cross reactivity

    Figure 4. 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 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 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.



    Figure 6. 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 6A: 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).

  • 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 H4K20me3 C15410057 DATASHEET
    Datasheet description
    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: H4K20me3 polyclonal antibody - Classic (Diagenode Cat# C15410057 Lot# A295-0014P). Click here to copy to clipboard.

    Using our products in your publication? Let us know!

    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 ...

    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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