Diagenode

H3K9me3 polyclonal antibody - Premium

Histone-Deacetylase-polyclonal-antibody-diagenode
Catalog Number
Format
Price
C15410193
(pAb-193-050)
50 μg/19 μl
(50 ChIP reactions)
$375.00
  Bulk order
Other format

Polyclonal antibody raised in rabbit against the region of histone H3 containing the trimethylated lysine 9 (H3K9me3), using a KLH-conjugated synthetic peptide.

LotA1671-001P
Concentration2.7 µg/µl
Species reactivityHuman, mouse, wide range expected
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 μg/ChIP Fig 1, 2
ELISA 1:5,000 Fig 3
Dot Blotting/Peptide array 1:20,000/1:2,000 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 figure 1
    ChIP figure 2

    Figure 1. ChIP results obtained with the Diagenode antibody directed against H3K9me3
    Figure 1A ChIP assays were performed using human HeLa cells, the Diagenode antibody against H3K9me3 (cat. No. pAb- 193-050) and optimized PCR primer pairs for qPCR. ChIP was performed with the ““Auto Histone ChIP-seq” kit (cat. No. AB- Auto02-A100) on The IP-Star automated system, using sheared chromatin from 1,000,000 cells. A titration consisting of 0.2, 0.5, 1, 2 and 5 μg of antibody per ChIP experiment was analyzed. IgG (1 μg/IP) was used as a negative IP control. Figure 1B ChIP assays were performed using human K562 cells, the Diagenode antibody against H3K9me3 (cat. No. pAb- 193-050) and optimized PCR primer pairs for qPCR. ChIP was performed with the “iDeal ChIP-seq” kit (cat. No. AB-001- 0024), using sheared chromatin from 100,000 cells. A titration consisting of 0.2, 0.5, 1 and 2 μg of antibody per ChIP experiment was analyzed. IgG (1 μg/IP) was used as a negative IP control. Quantitative PCR was performed with primers specific for the promoter of the active genes GAPDH, c-fos and EIF4A2, used as negative controls, and for ZNF510 and the Sat2 satellite repeat, used as positive controls. The figure 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

    Figure 2. ChIP-seq results obtained with the Diagenode antibody directed against H3K9me3
    ChIP was performed on sheared chromatin from 100,000 K562 cells using 1 μg of the Diagenode antibody against H3K9me3 (cat. No. pAb-193-050) with the iDeal ChIP-seq kit 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 36 bp tags were aligned to the human genome using the BWA algorithm. Figure 2A shows the H3K9me3 signal distribution along the long arm of human chromosome 19 and a zoomin to an enriched region containing several ZNF repeat genes. Figure 2B shows the signal distribution in a 200 kb region from chromosome 12 surrounding the ZNF12 gene.

    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 against H3K9me3 (cat. No. pAb-193-050). 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 1:115,000.

    Dot blot

    Figure 4. Cross reactivity tests using the Diagenode antibody directed against H3K9me3
    Figure 4A. To test the cross reactivity of the Diagenode antibody against H3K9me3 (cat.No. pAb-193-050), a Dot Blot analysis was performed with peptides containing other histone modifications and the unmodified H3K9. 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 1:2,000. 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 H3K9me3
    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 H3K9me3 (cat. No. pAb-193-050). 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 H3K9me3
    HeLa cells were stained with the Diagenode antibody against H3K9me3 (cat. No. pAb-193-050) 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 labeled with the H3K9me3 antibody (top) 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 at the bottom.

  • Testimonials

    In life sciences, epigenetics is nowadays the most rapid developing field with new astonishing discoveries made every day. To keep pace with this field, we are in need of reliable tools to foster our research - tools Diagenode provides us with. From antibodies to automated solutions - all from one source and with robust support. Antibodies used in our lab: H3K27me3 polyclonal antibody – Premium, H3K4me3 polyclonal antibody – Premium, H3K9me3 polyclonal antibody – Premium, H3K4me1 polyclonal antibody – Premium, CTCF polyclonal antibody – Classic, Rabbit IgG.

    Dr. Florian Uhle, Dept. of Anesthesiology, Heidelberg University Hospital, Germany
  • 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
    Peptide array
    Peptide array Read more
    ChIP-seq (ab)
    Read more
    ChIP-qPCR (ab)
    Read more
  • Documents
    Datasheet H3K9me3 pAb-193-050 DATASHEET
    Polyclonal antibody raised in rabbit against the region of histone H3 containing the trimethylate...
    Download
    Epigenetic Antibodies Brochure BROCHURE
    More than in any other immuoprecipitation assays, quality antibodies are critical tools in many e...
    Download
    Chromatin Immunoprecipitation Brochure BROCHURE
    Whether you are experienced or new to the field of chromatin immunoprecipitation, Diagenode has e...
    Download
    True MicroChIP and MicroPlex kits APPLICATION NOTE
    From minuscule amounts to magnificent results: reliable ChIP-seq data from 10,000 cells with the ...
    Download
    ChIP kit results with True MicroChIP kit POSTER
    Chromatin immunoprecipitation coupled with high-throughput sequencing (ChIP-seq) has become the g...
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    Antibodies you can trust POSTER
    Epigenetic research tools have evolved over time from endpoint PCR to qPCR to the analyses of lar...
    Download
  • Publications

    How to properly cite this product in your work

    Diagenode strongly recommends using this: H3K9me3 polyclonal antibody - Premium (Diagenode Cat# C15410193 Lot# A1671-001P). Click here to copy to clipboard.

    Using our products in your publication? Let us know!

    Neonatal monocytes exhibit a unique histone modification landscape
    Bermick JR et al.
    Background Neonates have dampened expression of pro-inflammatory cytokines and difficulty clearing pathogens. This makes them uniquely susceptible to infections, but the factors regulating neonatal-specific immune responses are poorly understood. Epigenetics, including histone modifications, can activate or silen...

    Epigenetic dynamics of monocyte-to-macrophage differentiation
    Wallner S et al.
    BACKGROUND: Monocyte-to-macrophage differentiation involves major biochemical and structural changes. In order to elucidate the role of gene regulatory changes during this process, we used high-throughput sequencing to analyze the complete transcriptome and epigenome of human monocytes that were differentiated in...

    Comprehensive genome and epigenome characterization of CHO cells in response to evolutionary pressures and over time
    Feichtinger J, Hernández I, Fischer C, Hanscho M, Auer N, Hackl M, Jadhav V, Baumann M, Krempl PM, Schmidl C, Farlik M, Schuster M, Merkel A, Sommer A, Heath S, Rico D, Bock C, Thallinger GG, Borth N
    The most striking characteristic of CHO cells is their adaptability, which enables efficient production of proteins as well as growth under a variety of culture conditions, but also results in genomic and phenotypic instability. To investigate the relative contribution of genomic and epigenetic modifications towards...

    Deciphering the principles that govern mutually exclusive expression of Plasmodium falciparum clag3 genes
    Rovira-Graells N, Crowley VM, Bancells C, Mira-Martínez S, de Pouplana LR, Cortés A
    The product of the Plasmodium falciparum genes clag3.1 and clag3.2 plays a fundamental role in malaria parasite biology by determining solute transport into infected erythrocytes. Expression of the two clag3 genes is mutually exclusive, such that a single parasite expresses only one of the two genes at a time. Here ...

    Epigenome mapping reveals distinct modes of gene regulation and widespread enhancer reprogramming by the oncogenic fusion protein EWS-FLI1.
    Tomazou EM, Sheffield NC, Schmidl C, Schuster M, Schönegger A, Datlinger P, Kubicek S, Bock C, Kovar H
    Transcription factor fusion proteins can transform cells by inducing global changes of the transcriptome, often creating a state of oncogene addiction. Here, we investigate the role of epigenetic mechanisms in this process, focusing on Ewing sarcoma cells that are dependent on the EWS-FLI1 fusion protein. We establi...

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