H4K20me3 Antibody
Polyclonal antibody raised in rabbit against the region of histone H4 containing the trimethylated lysine 20 (H4K20me3), using a KLH-conjugated synthetic peptide.
| Lot | A295-0014P |
|---|---|
| Concentration | 1.07 µg/µl |
| Species reactivity | Human, mouse |
| Type | Polyclonal |
| Purity | Affinity purified |
| Host | Rabbit |
| Precautions | This 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
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).
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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.
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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).
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.
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
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.
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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.
Applications
| ELISA Enzyme-linked immunosorbent assay. Read more |
| DB Dot blotting Read more |
| WB 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 |
| IF 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 The quality of antibody used in CUT&Tag is one of the crucial factors for assay success. The antibodies with confirmed high specificity will target only the protein of interest, enabling real results. Check out our selection of antibodies vali... Read more |
Documents
| Datasheet H4K20me3 C15410057 DATASHEET Datasheet description | Download |
| Antibodies you can trust POSTER Epigenetic research tools have evolved over time from endpoint PCR to qPCR to the analyses of lar... | Download |
| Epigenetic Antibodies Brochure BROCHURE More than in any other immuoprecipitation assays, quality antibodies are critical tools in many e... | Download |
Safety sheets
Publications
How to properly cite our product/service in your workWe strongly recommend using this: H4K20me3 Antibody (Hologic Diagenode Cat# C15410057 Lot# A295-0014P). Click here to copy to clipboard. Using our products or services in your publication? Let us know! |
Histone H4K20 tri-methylation at late-firing origins ensures timely heterochromatin replication |
Decoupling of DNA methylation and activity of intergenic LINE-1 promoters in colorectal cancer |
Heat shock represses rRNA synthesis by inactivation of TIF-IA and lncRNA-dependent changes in nucleosome positioning |
DOT1L Activity Promotes Proliferation and Protects Cortical Neural Stem Cells from Activation of ATF4-DDIT3-Mediated ER Stress In Vitro |
Use of a mouse in vitro fertilization model to understand the developmental origins of health and disease hypothesis. |
FSHD muscular dystrophy region gene 1 binds Suv4-20h1 histone methyltransferase and impairs myogenesis |
Expression of a large LINE-1-driven antisense RNA is linked to epigenetic silencing of the metastasis suppressor gene TFPI-2 in cancer. |
Overexpression of facioscapulohumeral muscular dystrophy region gene 1 causes primary defects in myogenic stem cells. |