Diagenode

H3K9/14ac polyclonal antibody - Premium (sample size)

Histone-Deacetylase-polyclonal-antibody-diagenode
Catalog Number
Format
Price
C15410200-10
10 µg
$80.00
  Bulk order
Other format

Polyclonal antibody raised in rabbit against the region of histone H3 containing the acetylated lysines 9 and 14 (H3K9/14ac), using a KLH-conjugated synthetic peptide. 

LotA381-004
Concentration1.39 µg/µl
Species reactivityHuman, mouse, zebrafish, Nematodes, A. Nidulans, Arabidopsis
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 1:100 Fig 3
Dot Blotting 1:20,000 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 1-5 μg per IP.

  • Validation Data

    ChIP result

    Figure 1. ChIP results obtained with the Diagenode antibody directed against H3K9/14ac
    ChIP assays were performed using human HeLa cells, the Diagenode antibody against H3K9/14ac (Cat. No. C15410200) 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 1, 2, 5 and 10 μg of antibody per ChIP experiment was analyzed. IgG (2 μg/IP) was used as a negative IP control. Quantitative PCR was performed with primers specific for the promoter of the active genes GAPDH and EIF4A2, used as positive controls, and for the coding region of the inactive MB gene and the Sat2 satellite repeat, used as negative 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

    Figure 2. ChIP-seq results obtained with the Diagenode antibody directed against H3K9/14ac
    ChIP was performed on sheared chromatin from 100,000 K562 cells using 1 μg the Diagenode antibody against H3K9/14ac (Cat. No. C15410200) with the “iDeal ChIP-seq” kit (Cat. No. AB-001-0024). IgG (1 μg/IP) was used as a negative IP control. The IP’d DNA was analysed by QPCR with optimized PCR primer pairs for the promoters of the active GAPDH and EIF4A2 genes, used as positive control targets, and the coding region of the inactive MB gene and the Sat2 satellite repeat, used as negative control targets (figure 2A). The IP’d DNA was subsequently analysed with 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 2 shows the peak distribution along the complete sequence and a 1.5 Mb region of the X-chromosome (figure 2B and C) and in two regions surrounding the GAPDH and EIF4A2 positive control genes, respectively (figure 2D and E). The position of the amplicon used for ChIP-qPCR is indicated by an arrow. These results clearly show an enrichment of the H3K9/14 acetylation at the promoters of active genes.

    ChIP-seq figure B

    ChIP-seq figure C

    ChIP-seq figure D

    ChIP-seq figure E

    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 H3K9/14ac (Cat. No. C15410200). 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 1:4,000.

    Cross reactivity figure A
    Cross reactivity figure B

    Figure 4. Cross reactivity tests using the Diagenode antibody directed against H3K9/14ac
    Figure 4A To test the cross reactivity of the Diagenode antibody against H3K9/14ac (Cat. No. C15410200), 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 H3K9/14ac
    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 H3K9/14ac (Cat. No. C15410200). The antibody was diluted 1:500 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 H3K9/14ac
    HeLa cells were stained with the Diagenode antibody against H3K9/14ac (Cat. No. C15410200) 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 H3K9/14ac antibody (left) diluted 1:200 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.

  • Testimonials

    The Diagenode Plant ChIP-seq kit works very well. The kit gives higher enrichment over background for the positive samples compared to our previous method. Using both the Plant ChIP-seq Kit and Diagenode’s Premium H3K9/14 ac polyclonal antibody, we performed ChIP-qPCR of H3K9/14 ac in region 3 of the TOC1 promoter. TOC1 is a circadian clock gene involved in evening loop that inhibits circadian clock genes expressed during the light phase of the day. We observed higher acetylation around TSS of TOC1 at the end of light phase in short day conditions, results that correlate with previously published data.

    Dorota Komar, Centre for Plant Biotechnology and Genomics, Madrid, Spain
  • 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 H3K914ac C15410200 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...
    Download
  • Publications

    How to properly cite this product in your work

    Diagenode strongly recommends using this: H3K9/14ac polyclonal antibody - Premium (sample size) (Diagenode Cat# C15410200-10 Lot# A381-004). Click here to copy to clipboard.

    Using our products in your publication? Let us know!

    H3.3 demarcates GC-rich coding and subtelomeric regions and serves as potential memory mark for virulence gene expression in Plasmodium falciparum
    Fraschka SA et al.
    Histones, by packaging and organizing the DNA into chromatin, serve as essential building blocks for eukaryotic life. The basic structure of the chromatin is established by four canonical histones (H2A, H2B, H3 and H4), while histone variants are more commonly utilized to alter the properties of specific chromatin d...

    Chromatin Immunoprecipitation Assay for the Identification of Arabidopsis Protein-DNA Interactions In Vivo
    Komar DN, Mouriz A, Jarillo JA, Piñeiro M
    Intricate gene regulatory networks orchestrate biological processes and developmental transitions in plants. Selective transcriptional activation and silencing of genes mediate the response of plants to environmental signals and developmental cues. Therefore, insights into the mechanisms that control plant gene expr...

    NAD(+)-SIRT1 control of H3K4 trimethylation through circadian deacetylation of MLL1.
    Aguilar-Arnal L, Katada S, Orozco-Solis R, Sassone-Corsi P
    The circadian clock controls the transcription of hundreds of genes through specific chromatin-remodeling events. The histone methyltransferase mixed-lineage leukemia 1 (MLL1) coordinates recruitment of CLOCK-BMAL1 activator complexes to chromatin, an event associated with cyclic trimethylation of histone H3 Lys4 (H...

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