H3K9ac Antibody
Polyclonal antibody raised in rabbit against histone H3, acetylated at lysine 9 (H3K9ac), using a KLH-conjugated synthetic peptide.
| Lot | A1645D |
|---|---|
| Concentration | 1 µg/µl |
| Species reactivity | Human, mouse, pig, zebrafish, Poplar, Daphnia, P. Falciparum: positive. Other species: not tested. |
| Type | Polyclonal, ChIP-grade, ChIP-seq grade, CUT&Tag-grade; |
| Purity | Affinity purified polyclonal antibody |
| Host | Rabbit |
| Storage Conditions | Store at -20°C; for long storage, store at -80°C. Avoid multiple freeze-thaw cycles. |
| Storage Buffer | PBS containing 0.02% azide and 0.02% 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 μg/ChIP | Fig 1, 2 |
| CUT&Tag | 0.2-0.5 μg | Fig 3 |
| ELISA | 1:1,000 | Fig 4 |
| Dot Blotting | 1:2,000 | Fig 5 |
| Western Blotting | 1:1,000 | Fig 6 |
| Immunofluorescence | 1:500 | Fig 7 |
* 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 H3K9ac
ChIP assays were performed using human HeLa cells, the antibody against H3K9ac (cat. No. C15410004) and optimized PCR primer pairs for qPCR. ChIP was performed with the iDeal ChIP-seq kit (cat. No. C01010051), using sheared chromatin from 500,000 cells. A titration consisting of 1, 2.5 and 5 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 and EIF4A2, used as positive controls, and for the inactive MYOD1 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). These results are in accordance with the observation that acetylation of K9 at histone H3 is associated with the promoters of active genes
Figure 2. ChIP-seq results obtained with the antibody directed against H3K9ac
ChIP was performed with 1 µg of the antibody against H3K9ac (cat. No. C15410004) as described above and 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 2 shows the peak distribution along the complete sequence and an 800 kb region of the X-chromosome (figure 2A and B) and in 100 kb regions surrounding the GAPDH and EIF4A2 positive control genes (figure 2C and D). These results clearly show an enrichment of H3K9ac at the promoters of active genes.
Figure 3. Cut&Tag results obtained with the antibody directed against H3K9ac
CUT&TAG was performed on 50,000 K562 cells using 0.2 µg of the Diagenode antibody against H3K9ac (cat. No. C15410004) and the Diagenode pA-Tn5 transposase (C01070001). The libraries were subsequently analysed on an Illumina NovaSeq sequencer (2x50 paired-end reads) according to the manufacturer’s instructions. The tags were aligned to the human genome (hg38) using the BWA algorithm. Figure 3 shows the peak distribution along the complete sequence and a 600 kb region of human chromosome 1 (figure 3A and B) and in 2 genomic regions surrounding the EIF2S3 gene on the X-chromosome and the ACTB gene on chromosome 7 (figure 3C and D, respectively).
Figure 4. Determination of the antibody titer
To determine the titer of the antibody, an ELISA was performed using a serial dilution of the antibody directed against H3K9ac (cat. No. C15410004) 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 4), the titer of the antibody was estimated to be 1:13,500.
Figure 5. Cross reactivity test using the antibody directed against H3K9ac
A Dot Blot analysis was performed to test the cross reactivity of the antibody against H3K9ac (cat. No. C15410004) with peptides containing other histone modifications and the unmodified H3K9 sequence. One hundred to 0.2 pmol of the respective peptides were spotted on a membrane. The antibody was used at a dilution of 1:2,000. Figure 5 shows a high specificity of the antibody for the modification of interest.
Figure 6. Western blot analysis using the antibody directed against H3K9ac
Histone extracts of HeLa cells (15 µg) were analysed by Western blot using the antibody against H3K9ac (cat. No. C15410004) 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 7. Immunofluorescence using the antibody directed against H3K9ac
HeLa cells were stained with the antibody against H3K9ac (cat. No. C15410004) 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 H3K9ac 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.
Testimonials
I have extensively used the antibodies against the histone modifications H3K4me3, H3k27me3, H3K9ac, H4k8ac and H3K18ac provided by Diagenode. The high level of specificity and selectivity of these antibodies in mouse brain samples, confirmed by using several negative and positive controls run in parallel with mouse brain tissue samples, ensured successful and reproducible results. I have been a Diagenode costumer for over one year now and I am extremely satisfied with the efficiency of the Bioruptor Pico for chromatin shearing as well as all of the ChIP materials (i.e., antibodies, blocking peptides, primer pairs for qPCR) provided by this company. Many thanks.
Dr. Ermelinda Lomazzo, Institute of Physiological Chemistry, AG Prof. Beat Lutz. University Medical Center of the Johannes Gutenberg University Mainz, 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 |
| ChIP-seq (ab) Read more |
| ChIP-qPCR (ab) Read more |
| CUT&Tag The quality of antibody used in CUT&Tag is one of the crucial factors for assay success. The antibodies with confirmed high specificity will target only the protein of interest, enabling real results. Check out our selection of antibodies vali... Read more |
Documents
| Datasheet H3K9ac C15410004 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: H3K9ac Antibody (Hologic Diagenode Cat# C15410004 Lot# A1645D). Click here to copy to clipboard. Using our products or services in your publication? Let us know! |
Endurance training increases a ubiquitylated form of histone H3 in the skeletal muscle, supporting Notch1 upregulation in an MDM2-dependent manner |
Noncanonical regulation of imprinted gene Igf2 by amyloid-beta 1-42 inAlzheimer's disease. |
Analyzing the Genome-Wide Distribution of Histone Marks byCUT\&Tag in Drosophila Embryos. |
The histone acetyltransferase KAT6A is recruited to unmethylatedCpG islands via a DNA binding winged helix domain. |
HISTONE DEACETYLASE 15 and MOS4-Associated Complex subunits3A/3B coregulate intron retention of ABA-responsive genes. |
Broad domains of histone marks in the highly compact macronucleargenome. |
CREBBP/EP300 acetyltransferase inhibition disrupts FOXA1-bound enhancers to inhibit the proliferation of ER+ breast cancer cells. |
Frataxin gene editing rescues Friedreich's ataxia pathology in dorsal rootganglia organoid-derived sensory neurons. |
Epigenetic, transcriptional and phenotypic responses in Daphnia magna exposed to low-level ionizing radiation |
An epigenetic map of malaria parasite development from host to vector. |
USP22-dependent HSP90AB1 expression promotes resistance to HSP90 inhibition in mammary and colorectal cancer. |
Impact of human sepsis on CCCTC-binding factor associated monocyte transcriptional response of Major Histocompatibility Complex II components. |
SIRT1-dependent epigenetic regulation of H3 and H4 histone acetylation in human breast cancer |
Epigenetic regulation of vascular NADPH oxidase expression and reactive oxygen species production by histone deacetylase-dependent mechanisms in experimental diabetes. |
Rapid Communication: The correlation between histone modifications and expression of key genes involved in accumulation of adipose tissue in the pig. |
Krüppel-like transcription factor KLF10 suppresses TGFβ-induced epithelial-to-mesenchymal transition via a negative feedback mechanism |
Lhx2 interacts with the NuRD complex and regulates cortical neuron subtype determinants Fezf2 and Sox11 |
Chronic stress leads to epigenetic dysregulation in the neuropeptide-Y and cannabinoid CB1 receptor genes in the mouse cingulate cortex |
H3K4 acetylation, H3K9 acetylation and H3K27 methylation in breast tumor molecular subtypes |
Epigenetic Modifications with DZNep, NaBu and SAHA in Luminal and Mesenchymal-like Breast Cancer Subtype Cells |
Molecular and Epigenetic Biomarkers in Luminal Androgen Receptor: A Triple Negative Breast Cancer Subtype |
Role of Annexin gene and its regulation during zebrafish caudal fin regeneration |
VEGF-mediated cell survival in non-small-cell lung cancer: implications for epigenetic targeting of VEGF receptors as a therapeutic approach |
Spatiotemporal control of estrogen-responsive transcription in ERα-positive breast cancer cells. |
Deciphering the principles that govern mutually exclusive expression of Plasmodium falciparum clag3 genes |
Dendritic cell development requires histone deacetylase activity. |
Lysine-specific demethylase 1 regulates differentiation onset and migration of trophoblast stem cells. |
IL-23 is pro-proliferative, epigenetically regulated and modulated by chemotherapy in non-small cell lung cancer. |
Histone tail acetylation in brain occurs in an unpredictable fashion after death. |
IL-20 is epigenetically regulated in NSCLC and down regulates the expression of VEGF. |
H3.5 is a novel hominid-specific histone H3 variant that is specifically expressed in the seminiferous tubules of human testes. |
Epigenetic Regulation of Glucose Transporters in Non-Small Cell Lung Cancer |
H2A.Z demarcates intergenic regions of the plasmodium falciparum epigenome that are dynamically marked by H3K9ac and H3K4me3. |
Histone modifications at the blastocyst Axin1(Fu) locus mark the heritability of in vitro culture-induced epigenetic alterations in mice. |
Plasmodium falciparum heterochromatin protein 1 marks genomic loci linked to phenotypic variation of exported virulence factors. |
A rapid micro chromatin immunoprecipitation assay (microChIP). |
Regulation of EP receptors in non-small cell lung cancer by epigenetic modifications. |