Diagenode

H4K20me3 polyclonal antibody - Premium

Histone-Deacetylase-polyclonal-antibody-diagenode

Catalog Number
Format
Price
C15410207
50 μg/53 μl
$375.00
Other format

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

LotA2730P
Concentration0.94 µg/µl
Species reactivityHuman, mouse, wide range: expected
TypePolyclonal
PurityAffinity purified
HostRabbit
Applications Suggested dilution References
ChIP * 0.5-1 μg per IP Fig 1, 2
ELISA 1:500 Fig 3
Dot Blotting/Peptide array 1:20,000/1:10,000 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 0.5-5 μg per IP.
  • Validation Data

    ChIP result

    Figure 1. ChIP results obtained with the Diagenode antibody directed against H4K20me3
    ChIP assays were performed using human K562 cells, the Diagenode antibody against H4K20me3 (Cat. No. C15410207) and optimized PCR primer sets for qPCR. ChIP was performed with the “iDeal ChIP- seq” kit (Cat. No. AB-001-0024) on sheared chromatin from 100,000 cells. A titration of the antibody consisting of 0.2, 0.5, 1 and 2 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 EIF4A2 and GAPDH, used as negative controls, and for ZNF510 and the Sat2 satellite repeat region used as positive 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 H4K20me3
    ChIP was performed with 0.5 μg of the Diagenode antibody against H4K20me3 (Cat. No. C15410207) on sheared chromatin from 100,000 K562 cells using the “iDeal ChIP-seq” kit. The IP’d DNA was analysed by QPCR as described above (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 in the telomeric region of chromosome 12, also containing several ZNF repeat genes, and at ZNF510 on chromosome 9, respectively. The position of the amplicon used for ChIP-qPCR is indicated by an arrow.

    ELISA

    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. C15410207) 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 1:21,700.

    Cross reactivity figure A

    Cross reactivity figure B

    B. Figure 4. Cross reactivity tests using the Diagenode antibody directed against H4K20me3
    Figure 4A To test the cross reactivity of the Diagenode antibody against H4K20me3 (Cat. No. C15410207), a Dot Blot analysis was performed 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 4A shows a high specificity of the antibody for the modification of interest. Figure 4B The specificity of the antibody was further demonstrated by peptide array analyses on an array containing 384 peptides with different combinations of modifications from histone H3, H4, H2A and H2B. The antibody was used at a dilution of 1:10,000. Figure 4B shows the specificity factor, calculated as the ratio of the average intensity of all spots containing the mark, divided by the average intensity of all spots not containing the mark.

    Western blot

    Figure 5. Western blot analysis using the Diagenode antibody directed against H4K20me3
    Western blot was performed on whole cell (25 μg, lane 1) and histone extracts (15 μg, lane 2) from HeLa cells, and on 1 μg of recombinant histone H2A, H2B, H3 and H4 (lane 3, 4, 5 and 6, respectively) using the Diagenode antibody against H4K20me3 (Cat. No. C15410207). 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.

    Immunofluorescence

    Figure 6. Immunofluorescence using the Diagenode antibody directed against H4K20me3
    HeLa cells were stained with the Diagenode antibody against H4K20me3 (Cat. No. C15410207) and with DAPI. Cells were fixed with methanol and blocked with PBS/TX-100 containing 5% normal goat serum and 1% BSA. The cells were immunofluorescently labeled with the H4K20me3 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
    ELISA
    Enzyme-linked immunosorbent assay. Read more
    DB
    Dot blotting Read more
    Peptide array
    Peptide array 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
  • Documents
    Datasheet H4K20me3 C15410207 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...
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    Epigenetic Antibodies Brochure BROCHURE
    More than in any other immuoprecipitation assays, quality antibodies are critical tools in many e...
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