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H3K4me3 Antibody - ChIP-seq Grade (sample size)

Catalog Number
Format
Price
C15410030-10
10 µg
$97.00
  Bulk order
Other format



Polyclonal antibody raised in rabbit against the region of histone H3 containing the trimethylated lysine 4 (H3K4me3), using a KLH-conjugated synthetic peptide.

Lot002
Concentration1.4 µg/µl
Species reactivityHuman, mouse, Arabidopsis: positive. Other species: not tested.
TypePolyclonal
PurityAffinity purified polyclonal antibody.
HostRabbit
Storage ConditionsStore at -20°C; for long storage, store at -80°C. Avoid multiple freeze-thaw cycles.
Storage BufferPBS containing 0.05% azide and 0.05% ProClin 300.
PrecautionsThis product is for research use only. Not for use in diagnostic or therapeutic procedures.
Applications Suggested dilution References
ChIP/ChIP-seq * 1 µg/ChIP Fig 1, 2
Dot Blotting 1:2,000 Fig 3
Western Blotting 1:500 Fig 4
Immunofluorescence 1:100 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

    A. H3K4me3 Antibody ChIP Grade

    B. H3K4me3 Antibody ChIP Grade

    Figure 1. ChIP results obtained with the Diagenode antibody directed against H3K4me3
    ChIP assays were performed using human HeLa cells, the Diagenode antibody against H3K4me3 (Cat. No. C15410030) and optimized PCR primer pairs for qPCR. ChIP was performed with the “iDeal ChIP-seq” kit (cat. No. C01010051), using sheared chromatin from 1 million cells. The chromatin was spiked with a panel of in vitro assembled nucleosomes, each containing a specific lysine methylation (SNAP-ChIP K-MetStat Panel, Epicypher). A titration consisting of 1, 2, 5 and 10 µg of antibody per ChIP experiment was analyzed. IgG (2 µg/IP) was used as a negative IP control. Figure 1A. Quantitative PCR was performed with primers specific for the promoter of the active GAPDH and EIF4A2 genes, used as positive controls, and for the inactive MYOD1 gene, used as negative control. The graph 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 trimethylation of K4 at histone H3 is associated with the promoters of active genes Figure 1B. Recovery of the nucleosomes carrying the H3K4me1, H3K4me2, H3K4me3 modifications and the unmodified H3K4 as determined by qPCR. The figure clearly shows the antibody is very specific in ChIP for the H3K4me3 modification. At higher concentrations some H3K4me2 is also precipitated.

    A. H3K4me3 Antibody ChIP-seq Grade

    B. H3K4me3 Antibody for ChIP-seq

    C. H3K4me3 Antibody for ChIP-seq assay

    D. H3K4me3 Antibody validated in ChIP-seq

    Figure 2. ChIP-seq results obtained with the Diagenode antibody directed against H3K4me3
    ChIP was performed on sheared chromatin from 1 million HeLa cells using 1 µg of the Diagenode antibody against H3K4me3 (Cat. No. C15410030) as described above. 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 50 bp tags were aligned to the human genome using the BWA algorithm. Figure 2 shows the peak distribution along the complete sequence and a 1.2 Mb region of the X-chromosome (figure 2A and B) and in two regions surrounding the GAPDH and EIF4A2 positive control genes, respectively (figure 2C and D). These results clearly show an enrichment of the H3K4 trimethylation at the promoters of active genes.

    H3K4me3 Antibody validated in Dot Blot

    Figure 3. Cross reactivity test using the Diagenode antibody directed against H3K4me3
    A Dot Blot analysis was performed to test the cross reactivity of the Diagenode antibody against H3K4me3 (Cat. No. C15410030) with peptides containing other modifications and unmodified sequences of histone H3 and H4. One hundred to 0.2 pmol of the respective peptides 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.

    H3K4me3 Antibody validated in Western Blot

    Figure 4. Western blot analysis using the Diagenode antibody directed against H3K4me3
    Western blot was performed on whole cell (40 µg, lane 1) and histone extracts (15 µg, lane 2) from HeLa cells, and on 1 µg of recombinant histone H2A, H2B, H3.1, H3.3 and H4 (lane 3, 4, 5, 6 and 7, respectively) using the Diagenode antibody against H3K4me3 (Cat. No. C15410030). The antibody was diluted 1:500 in TBS-Tween containing 5% skimmed milk. The position of the protein of interest is shown on the right, the marker (in kDa) is shown on the left.

    H3K4me3 Antibody validated in Immunofluorescence

    Figure 5. Immunofluorescence using the Diagenode antibody directed against H3K4me3
    HeLa cells were stained with the Diagenode antibody against H3K4me3 (Cat. No. C15410030) and with DAPI. Cells were fixed with 4% formaldehyde for 10’ and blocked with PBS/TX-100 containing 5% normal goat serum and 1% BSA. The cells were immunofluorescently labelled with the H3K4me3 antibody (left) diluted 1:100 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.

  • 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. Trimethylation of histone H3K4 is associated with active promoters.

  •  Applications
    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
    DB
    Dot blotting 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
    Epigenetic Antibodies Brochure BROCHURE
    More than in any other immuoprecipitation assays, quality antibodies are critical tools in many e...
    Download
    Datasheet H3K4me3 C15410030 DATASHEET
    Datasheet description
    Download
  •  Publications

    How to properly cite this product in your work

    Diagenode strongly recommends using this: H3K4me3 Antibody - ChIP-seq Grade (sample size) (Diagenode Cat# C15410030-10 Lot# 002). Click here to copy to clipboard.

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