Diagenode

H3K27me3S28p polyclonal antibody - Classic

Histone-Deacetylase-polyclonal-antibody-diagenode
Catalog Number
Format
Price
C15410091
50 μg/ 295 μl
$295.00
  Bulk order

Polyclonal antibody raised in rabbit against against histone H3, trimethylated at lysine 27 and phosphorylated at serine 28 (H3K27me3S28p), using a KLH-conjugated synthetic peptide.

LotA154-0011
Concentration0.17 µg/µl
Species reactivityHuman
TypePolyclonal
PurityAffinity purified
HostRabbit
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 1:200 Fig 3
Dot Blotting 1:2,000 Fig 4
Western Blotting 1:1,000 Fig 5
* 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

    ChIP

    Figure 1. ChIP results obtained with the Diagenode antibody directed against H3K27me3S28p
    ChIP assays were performed using human HeLa cells treated with colcemid, the Diagenode antibody against H3K27me3S28p (Cat. No. C15410091) and optimized PCR primer sets for qPCR. ChIP was performed with the “iDeal ChIP-seq” kit (Cat. No. C01010051), using sheared chromatin from 1 million cells. 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 promoter of the active gene GAPDH as a negative control, and for the coding regions of the inactive genes MYT1 and TSH2B 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). These results are in accordance with the observation that H3K27me3S28p is preferably present at inactive genes.

    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 H3K27me3S28p
    ChIP was performed on sheared chromatin from 1.5 million colcemid treated HeLaS3 cells using 2 μg of the Diagenode antibody against H3K27me3S28p (Cat. No. C15410091) as described above. The IP’d DNA was subsequently analysed on an Illumina HiSeq. Library preparation, cluster generation and sequencing were performed according to the manufacturer’s instructions. The 51 bp tags were aligned to the human genome using the BWA algorithm. Figure 2 shows the enrichment in genomic regions surrounding the TSH2B and MYT1 positive control genes (fig 2A and B, indicated by an arrow), and of chromosome 3 and 20 (figure 2C and D).

    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 H3K27me3S28p (Cat. No. C15410091). The antigen used was a peptide containing the histone modification of interest. By plotting the absorbance against the antibody dilution (Figure 2), the titer of the antibody was estimated to be 1:4,300.

    Dot Blot

    Figure 4. Cross reactivity test of the Diagenode antibody directed against H3K27me3S28p
    A Dot Blot analysis was performed to test the cross reactivity of the Diagenode antibody against H3K27me3S28p (Cat. No. C15410091) with peptides containing other modifications of histone H3 and H4 and the unmodified H3K27S28 sequence. 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 1:2,000. Figure 3 shows a high specificity of the antibody for the modification of interest.

    Western blot figure A

    Western blot figure B

    Figure 5. Western blot analysis using the Diagenode antibody directed against H3K27me3S28p
    A. Western blot was performed on histone extracts (15 μg, lane 1) from HeLa cells, and on 1 μg of recombinant histone H3 (lane 2) using the Diagenode antibody against H3K27me3S28p (Cat. No. C15410091) diluted 1:100 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. B. Cell cycle experiment using the Diagenode antibody against H3K27me3S28p. WB was performed on cells blocked in different stages of the cell cycle (1=asynchronous population, 2=G1/Sa, 3=G1/Sb, 4=S phase, 5=G2/M, 6=M, 7=G1). The picture shows a nice peak of H3K27me3S28p expression during mitosis.

  • 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
    ChIP-seq (ab)
    Read more
    ChIP-qPCR (ab)
    Read more
  • Documents
    Datasheet H3K27me3S28p C15410091 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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  • Publications

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  • Workshop on Chromatin Proteomics
    Crete, Greece
    Oct 3-Oct 8, 2016
  • 2nd Annual Next Generation Sequencing Congress
    Boston, USA
    Oct 3-Oct 4, 2016
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