Diagenode

H3K27ac polyclonal antibody - Classic

Histone-Deacetylase-polyclonal-antibody-diagenode
Catalog Number
Format
Price
C15410174
(pAb-174-050)
50 μg/42 μl
$295.00
  Bulk order
Other format

Polyclonal antibody raised in rabbit against histone H3, acetylated at lysine 27 (H3K27ac), using a KLH-conjugated synthetic peptide.

LotA7071-001P
Concentration1.2 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/ChIP-seq * 1-2 μg/ChIP Fig 1, 2
ELISA 1:100 Fig 3
Dot Blotting 1:25,000 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

    Figure 1. ChIP results obtained with the Diagenode antibody directed against H3K27ac
    ChIP assays were performed using HeLa cells, the Diagenode antibody against H3K27ac (Cat. No. C15410174) and optimized PCR primer sets for qPCR. ChIP was performed with the “Auto Histone ChIP-seq” kit (Cat. No. C01010022), 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. Quantitative PCR was performed with primers for the promoter of the active ACTB (Cat. No. pp-1005-050) and EIF4A2 genes, used as positive controls, and for the coding region of the inactive MYT1 and TSH2B genes, 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). These results are in accordance with the observation that H3K27 acetylation is enriched at the promoters of active genes.

    ChIP-seq figure A

    Figure 2. ChIP-seq results obtained with the Diagenode antibody directed against H3K27ac
    ChIP was performed on sheared chromatin from 1 million HeLaS3 cells using 2 μg of the Diagenode antibody against H3K27ac (Cat. No. C15410174) as described above. Quantitative PCR was performed with primers for the promoter of the active ACTB (Cat. No. pp-1005-050) and EIF4A2 genes, used as positive controls, and for the coding region of the inactive MYT1 and MB (Cat. No. C17011006) genes, used as negative controls (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 peak distribution along the complete X-chromosome and in two regions surrounding the ACTB and EIF4A2 positive control genes, respectively (figure 2C and D).

    ChIP-seq figure B

    ChIP-seq figure C

    ChIP-seq figure 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 H3K27ac (Cat. No. C15410174) 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 1:4,000.

    Dot blot

    Figure 4. Western blot analysis using the Diagenode antibody directed against H3K27ac
    A Dot Blot analysis was performed to test the cross reactivity of the Diagenode antibody against H3K27ac (Cat. No. C15410174) with peptides containing other histone modifications and the unmodified H3K27 sequence. One hundred to 0.2 pmol of the respective peptides were spotted on a membrane. The antibody was used at a dilution of 1:25,000. Figure 4 shows a high specificity of the antibody for the modification of interest.

    Western blot

    Figure 5. Western blot analysis using the Diagenode antibody directed against H3K27ac
    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 and H4 (lane 3, 4, 5 and 6, respectively) using the Diagenode antibody against H3K27ac (Cat. No. C15410174). 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.

    Immunofloorescence

    Figure 6. Immunofluorescence using the Diagenode antibody directed against H3K27ac
    HeLa cells were stained with the Diagenode antibody against H3K27ac (Cat. No. C15410174) 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 H3K27ac 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
    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 H3K27ac pAb-174-050 DATASHEET
    Polyclonal antibody raised in rabbit against histone H3, acetylated at lysine 27 (H3K27ac), using...
    Download
    Epigenetic Antibodies Brochure BROCHURE
    More than in any other immuoprecipitation assays, quality antibodies are critical tools in many e...
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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...
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  • Publications

    How to properly cite this product in your work

    Diagenode strongly recommends using this: H3K27ac polyclonal antibody - Classic (Diagenode Cat# C15410174 Lot# A7071-001P). Click here to copy to clipboard.

    Using our products in your publication? Let us know!

    Phenotypic Plasticity through Transcriptional Regulation of the Evolutionary Hotspot Gene tan in Drosophila melanogaster
    Gibert JM et al.
    Phenotypic plasticity is the ability of a given genotype to produce different phenotypes in response to distinct environmental conditions. Phenotypic plasticity can be adaptive. Furthermore, it is thought to facilitate evolution. Although phenotypic plasticity is a widespread phenomenon, its molecular mechanisms are...

    Premalignant SOX2 overexpression in the fallopian tubes of ovarian cancer patients: Discovery and validation studies
    Hellner K et al.
    Current screening methods for ovarian cancer can only detect advanced disease. Earlier detection has proved difficult because the molecular precursors involved in the natural history of the disease are unknown. To identify early driver mutations in ovarian cancer cells, we used dense whole genome sequencing of micro...

    Genome-wide p63-regulated gene expression in differentiating epidermal keratinocytes
    Otia M, Kouwenhovena EN, Zhou H
    The transcription factor p63 is a key regulator in epidermal keratinocyte proliferation and differentiation. However, the role of p63 in gene regulation during these processes is not well understood. To investigate this, we recently generated genome-wide profiles of gene expression, p63 binding sites and active regu...

    Spatiotemporal control of estrogen-responsive transcription in ERα-positive breast cancer cells.
    P-Y Hsu, H-K Hsu, T-H Hsiao, Z Ye, E Wang, A L Profit, I Jatoi, Y Chen, N B Kirma, V X Jin, Z D Sharp and T H-M Huang
    Recruitment of transcription machinery to target promoters for aberrant gene expression has been well studied, but underlying control directed by distant-acting enhancers remains unclear in cancer development. Our previous study demonstrated that distant estrogen response elements (DEREs) located on chromosome 20q13...

    Transcription factor p63 bookmarks and regulates dynamic enhancers during epidermal differentiation
    Kouwenhoven EN, Oti M, Niehues H, van Heeringen SJ, Schalkwijk J, Stunnenberg HG, van Bokhoven H, Zhou H
    The transcription factor p63 plays a pivotal role in keratinocyte proliferation and differentiation in the epidermis. However, how p63 regulates epidermal genes during differentiation is not yet clear. Using epigenome profiling of differentiating human primary epidermal keratinocytes, we characterized a catalog of d...

    The Activation of IL-1-Induced Enhancers Depends on TAK1 Kinase Activity and NF-κB p65.
    Jurida L, Soelch J, Bartkuhn M, Handschick K, Müller H, Newel D, Weber A, Dittrich-Breiholz O, Schneider H, Bhuju S, Saul VV, Schmitz ML, Kracht M
    The inflammatory gene response requires activation of the protein kinase TAK1, but it is currently unknown how TAK1-derived signals coordinate transcriptional programs in the genome. We determined the genome-wide binding of the TAK1-controlled NF-κB subunit p65 in relation to active enhancers and promoters of transc...

    Nitric oxide-induced neuronal to glial lineage fate-change depends on NRSF/REST function in neural progenitor cells.
    Bergsland M, Covacu R, Perez Estrada C, Svensson M, Brundin L
    Degeneration of CNS tissue commonly occurs during neuroinflammatory conditions, such as multiple sclerosis (MS) and neurotrauma. During such conditions, neural stem/progenitor cell (NPC) populations have been suggested to provide new cells to degenerated areas. In the normal brain, NPCs from the SVZ generate neurons...

    A novel microscopy-based high-throughput screening method to identify proteins that regulate global histone modification levels.
    Baas R, Lelieveld D, van Teeffelen H, Lijnzaad P, Castelijns B, van Schaik FM, Vermeulen M, Egan DA, Timmers HT, de Graaf P
    Posttranslational modifications of histones play an important role in the regulation of gene expression and chromatin structure in eukaryotes. The balance between chromatin factors depositing (writers) and removing (erasers) histone marks regulates the steady-state levels of chromatin modifications. Here we describe...

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