H3K9/14ac Antibody
Polyclonal antibody raised in rabbit against histone H3 acetylated at lysines 9 and 14 (H3K9/14ac), using a KLH-conjugated synthetic peptide.
| Lot | A381-004 |
|---|---|
| Concentration | 1.39 µg/µl |
| Species reactivity | Human, mouse, zebrafish, Nematodes, A. Nidulans, Arabidopsis: positive. Other species: not tested. |
| Type | Polyclonal, ChIP-grade, ChIP-seq grade |
| Purity | Affinity purified |
| Host | Rabbit |
| Storage Conditions | Store at -20°C; for long storage, store at -80°C. Avoid multiple freeze-thaw cycles. |
| Storage Buffer | PBS containing 0.05% azide and 0.05% ProClin 300. |
| Precautions | This 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 | 1:5,000-1:10,000 | 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
Figure 1.ChIP results obtained with the antibody directed against H3K9/14ac
ChIP assays were performed using HeLa cells, the antibody against H3K9/14ac (cat. No. C15410005) and optimized primer pairs for qPCR. ChIP was performed on sheared chromatin from 1.5 million cells. A titration of the antibody consisting of 1, 2, 5 and 10 µg per ChIP experiment was analysed. IgG (5 µg/IP) was used as a negative IP control. qPCR was performed using primers specific for the promoter of the ACTB gene (cat. No. C17011005) as a positive control target and for exon 2 of the MB gene (cat. No. C17011006) as a negative control target. Figure 1 shows the recovery (the relative amount of immunoprecipitated DNA compared to input DNA). These results confirm the observation that acetylation of H3K9/14 is present at active promoters.
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Figure 2. ChIP-seq results obtained with the antibody directed against H3K9/14ac
ChIP was performed with 1 µg of the antibody against H3K9/14ac (cat. No. C15410005) on sheared chromatin from 1 million HeLaS3 cells. IgG (2 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 c-fos 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 an 800 kb region of the X-chromosome (figure 2B and C) and in 100 kb regions surrounding the RBM3, GAPDH and c-fos genes (figure 2D, E and F). These results clearly show an enrichment of the H3K9/14 double acetylation at the promoters of active genes.
Figure 3. Determination of the titer
To determine the titer of the antibody, an ELISA was performed using a serial dilution of the antibody directed against H3K9/14ac (cat. No. C15410005) 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 purified antibody was estimated to be 1:65,500.
Figure 4. Cross reactivity test using the antibody directed against H3K9/14ac
A Dot Blot analysis was performed to test the cross reactivity of the antibody against H3K9/14ac (cat. No. C15410005) with peptides containing other histone modifications and the unmodified H3K9/14 sequence. One hundred to 0.2 pmol of the respective peptides were spotted on a membrane. The antibody was used at a dilution of 1:20,000. Figure 4 shows a high specificity of the antibody for the modification of interest.
Figure 5. Western blot analysis using the antibody directed against H3K9/14ac
Histone extracts of HeLa cells (15 µg) were analysed by Western blot using the antibody directed against H3K9/14ac (cat. No. C15410005) 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.
Figure 6. Immunofluorescence using the antibody directed against H3K9/14ac
Mouse NIH3T3 cells were stained with the antibody against H3K9/14ac (cat. No. C15410005) 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 H3K9/14ac 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.
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. Acetylation of H3K9/14 is enriched near the promoters of active genes.active genes.
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 H3K914ac C15410005 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... | Download |
| Epigenetic Antibodies Brochure BROCHURE More than in any other immuoprecipitation assays, quality antibodies are critical tools in many e... | Download |
Safety sheets
Publications
How to properly cite our product/service in your workWe strongly recommend using this: H3K9/14ac Antibody (Hologic Diagenode Cat# C15410005 Lot# A381-004). Click here to copy to clipboard. Using our products or services in your publication? Let us know! |
Temporal modification of H3K9/14ac and H3K4me3 histone marksmediates mechano-responsive gene expression during the accommodationprocess in poplar |
Efficacy of selective histone deacetylase 6 inhibition in mouse models ofPseudomonas aeruginosa infection: A new glimpse for reducinginflammation and infection in cystic fibrosis. |
Histone demethylase KDM2A suppresses EGF-TSPAN8 pathway toinhibit breast cancer cell migration and invasion in vitro. |
Transferrin Receptor 1 Regulates Thermogenic Capacity and Cell Fate in Brown/Beige Adipocytes |
Alteration of CTCF-associated chromatin neighborhood inhibits TAL1-driven oncogenic transcription program and leukemogenesis. |
Integrative Proteomic Profiling Reveals PRC2-Dependent Epigenetic Crosstalk Maintains Ground-State Pluripotency. |
The Hematopoietic Transcription Factors RUNX1 and ERG Prevent AML1-ETO Oncogene Overexpression and Onset of the Apoptosis Program in t(8;21) AMLs |
Role of Annexin gene and its regulation during zebrafish caudal fin regeneration |
Germline organization in Strongyloides nematodes reveals alternative differentiation and regulation mechanisms. |
VEGF-mediated cell survival in non-small-cell lung cancer: implications for epigenetic targeting of VEGF receptors as a therapeutic approach |
The oncofusion protein FUS-ERG targets key hematopoietic regulators and modulates the all-trans retinoic acid signaling pathway in t(16;21) acute myeloid leukemia. |
Membrane-Bound Methyltransferase Complex VapA-VipC-VapB Guides Epigenetic Control of Fungal Development. |
Histone deacetylase complex1 expression level titrates plant growth and abscisic Acid sensitivity in Arabidopsis. |
IL-23 is pro-proliferative, epigenetically regulated and modulated by chemotherapy in non-small cell lung cancer. |
ERG and FLI1 binding sites demarcate targets for aberrant epigenetic regulation by AML1-ETO in acute myeloid leukemia. |
Pre-B cell to macrophage transdifferentiation without significant promoter DNA methylation changes. |
IL-20 is epigenetically regulated in NSCLC and down regulates the expression of VEGF. |
Epigenetic Regulation of Glucose Transporters in Non-Small Cell Lung Cancer |
Epigenetic control of vascular smooth muscle cells in Marfan and non-Marfan thoracic aortic aneurysms. |
Regulation of EP receptors in non-small cell lung cancer by epigenetic modifications. |