H3K9me3 Antibody (sample size)

Lot	A0219P
Concentration	0.9 µg/µl
Species reactivity	Human, mouse, yeast, wide range expected.
Type	Polyclonal, ChIP grade, ChIP-seq 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 0.05% 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 µg/ChIP	Fig 1, 2
ELISA	1:1000	Fig 3
Dot Blotting/Peptide array	1:20,000/1:5,000	Fig 4
Western Blotting	1:1,000	Fig 5
Immunofluorescence	1:250	Fig 6

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Figure 1. ChIP results obtained with the Diagenode antibody directed against H3K9me3
ChIP assays were performed using human HeLa cells, the Diagenode antibody against H3K9me3 (Cat. No. C15410193) and optimized PCR primer pairs for qPCR. ChIP was performed with the "iDeal ChIP-seq" kit (Cat. No. C01010051), 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 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).

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Figure 2. ChIP-seq results obtained with the Diagenode antibody directed against H3K9me3
ChIP was performed with 1 μg of the Diagenode antibody against H3K9me3 (Cat. No. C15410193) on sheared chromatin from 1,000,000 HeLa cells using the “iDeal ChIP-seq” kit as described above. The IP'd DNA was subsequently analysed on an Illumina HiSeq 2000. Library preparation, cluster generation and sequencing were performed according to the manufacturer's instructions. The 50 bp tags were aligned to the human genome using the BWA algorithm. Figure 2A shows the signal distribution along the long arm of chromosome 19 and a zoomin to an enriched region containing several ZNF repeat genes. The arrows indicate two satellite repeat regions which exhibit a stronger signal. Figure 2C and D show the enrichment at the KCNQ1 and H19 imprinted genes.

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. C15410193). 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 1:42,700.

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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. C15410193), 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 1:5,000. 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.

Figure 5. Western blot analysis using the Diagenode antibody directed against H3K9me3
Western blot was performed on whole cell (50 μg, lane 1) and histone extracts (15 μg, lane 2) from HeLa cells, and on 1 μg of recombinant histone H2A, H2B, H3.1, H3.2 and H4 (lane 3, 4, 5, 6 and 7, respectively) using the Diagenode antibody against H3K9me3 (Cat. No. C15410193). The antibody was diluted 1:1,000 in TBSTween 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 Diagenode antibody directed against H3K9me3
HeLa cells were stained with the Diagenode antibody against H3K9me3 (Cat. No. C15410193) 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 (left) diluted 1:250 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. Trimethylation of histone H3K9 is associated with inactive genomic regions, satellite repeats and ZNF gene repeats.

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

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

ELISA
Enzyme-linked immunosorbent assay. Read more

DB
Dot blotting 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

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
Datasheet H3K9me3 pAb-193-050 DATASHEET Polyclonal antibody raised in rabbit against the region of histone H3 containing the trimethylate...	Download

Publications

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The senolytic cocktail, dasatinib and quercetin, impacts the chromatin structure of both young and senescent vascular smooth muscle cells
Agnieszka Gadecka et al.
One promising strategy to alleviate aging symptoms is the treatment with senolytics that is compounds which selectively eliminate senescent cells. Some therapies aim to reduce symptoms of cellular senescence without senescent cell eradication (senomorphic activity). However, senotherapies raise many questions concer...

MECOM is a master repressor of myeloid differentiation through dose control of CEBPA in acute myeloid leukemia
Dorien Pastoors et al.
Enhancer translocations, due to 3q26 rearrangements, drive out-of-context MECOM expression in an aggressive subtype of acute myeloid leukemia (AML). Direct depletion of MECOM using an endogenous auxin-inducible degron immediately upregulates expression of myeloid differentiation factor CEBPA. MECOM depletion is also...

Nuclear localization of MTHFD2 is required for correct mitosis progression
Natalia Pardo-Lorente et al.
Subcellular compartmentalization of metabolic enzymes establishes a unique metabolic environment that elicits specific cellular functions. Indeed, the nuclear translocation of certain metabolic enzymes is required for epigenetic regulation and gene expression control. Here, we show that the nuclear localization of t...

Claudin-1 as a potential marker of stress-induced premature senescence in vascular smooth muscle cells
Agnieszka Gadecka et al.
Cellular senescence, a permanent state of cell cycle arrest, can result either from external stress and is then called stress-induced premature senescence (SIPS), or from the exhaustion of cell division potential giving rise to replicative senescence (RS). Despite numerous biomarkers distinguishing SIPS from RS rema...

RNA sequestration in P-bodies sustains myeloid leukaemia
Srikanth Kodali et al.
Post-transcriptional mechanisms are fundamental safeguards of progenitor cell identity and are often dysregulated in cancer. Here, we identified regulators of P-bodies as crucial vulnerabilities in acute myeloid leukaemia (AML) through genome-wide CRISPR screens in normal and malignant haematopoietic progenitors. We...

A multiomic atlas of the aging hippocampus reveals molecular changes in response to environmental enrichment
Perez R. F. at al.
Aging involves the deterioration of organismal function, leading to the emergence of multiple pathologies. Environmental stimuli, including lifestyle, can influence the trajectory of this process and may be used as tools in the pursuit of healthy aging. To evaluate the role of epigenetic mechanisms in this context, ...

In vitro production of cat-restricted Toxoplasma pre-sexual stages
Antunes, A.V. et al.
Sexual reproduction of Toxoplasma gondii, confined to the felid gut, remains largely uncharted owing to ethical concerns regarding the use of cats as model organisms. Chromatin modifiers dictate the developmental fate of the parasite during its multistage life cycle, but their targeting to stage-specific cistro...

Alterations in the hepatocyte epigenetic landscape in steatosis.
Maji Ranjan K. et al.
Fatty liver disease or the accumulation of fat in the liver, has been reported to affect the global population. This comes with an increased risk for the development of fibrosis, cirrhosis, and hepatocellular carcinoma. Yet, little is known about the effects of a diet containing high fat and alcohol towards epigenet...

Chromatin profiling identifies transcriptional readthrough as a conservedmechanism for piRNA biogenesis in mosquitoes.
Qu J. et al.
The piRNA pathway in mosquitoes differs substantially from other model organisms, with an expanded PIWI gene family and functions in antiviral defense. Here, we define core piRNA clusters as genomic loci that show ubiquitous piRNA expression in both somatic and germline tissues. These core piRNA clusters are enriche...

Epigenetic dosage identifies two major and functionally distinct beta cells ubtypes.
Dror E.et al.
The mechanisms that specify and stabilize cell subtypes remain poorly understood. Here, we identify two major subtypes of pancreatic Î² cells based on histone mark heterogeneity (beta HI and beta LO). Beta HI cells exhibit 4-fold higher levels of H3K27me3, distinct chromatin organization and compaction, a...

Species-specific regulation of XIST by the JPX/FTX orthologs.
Rosspopoff O. et al.
X chromosome inactivation (XCI) is an essential process, yet it initiates with remarkable diversity in various mammalian species. XIST, the main trigger of XCI, is controlled in the mouse by an interplay of lncRNA genes (LRGs), some of which evolved concomitantly to XIST and have orthologues across all placental mam...

Noncanonical regulation of imprinted gene Igf2 by amyloid-beta 1-42 inAlzheimer's disease.
Fertan E. et al.
Reduced insulin-like growth factor 2 (IGF2) levels in Alzheimer's disease (AD) may be the mechanism relating age-related metabolic disorders to dementia. Since Igf2 is an imprinted gene, we examined age and sex differences in the relationship between amyloid-beta 1-42 (Aβ) accumulation and epigenetic regulation...

Histone remodeling reflects conserved mechanisms of bovine and humanpreimplantation development.
Zhou C. et al.
How histone modifications regulate changes in gene expression during preimplantation development in any species remains poorly understood. Using CUT\&Tag to overcome limiting amounts of biological material, we profiled two activating (H3K4me3 and H3K27ac) and two repressive (H3K9me3 and H3K27me3) marks in bovine...

Dietary methionine starvation impairs acute myeloid leukemia progression.
Cunningham A. et al.
Targeting altered tumor cell metabolism might provide an attractive opportunity for patients with acute myeloid leukemia (AML). An amino acid dropout screen on primary leukemic stem cells and progenitor populations revealed a number of amino acid dependencies, of which methionine was one of the strongest. By using v...

Dominant role of DNA methylation over H3K9me3 for IAP silencingin endoderm.
Wang Z. et al.
Silencing of endogenous retroviruses (ERVs) is largely mediated by repressive chromatin modifications H3K9me3 and DNA methylation. On ERVs, these modifications are mainly deposited by the histone methyltransferase Setdb1 and by the maintenance DNA methyltransferase Dnmt1. Knock-out of either Setdb1 or Dnmt1 leads to...

bESCs from cloned embryos do not retain transcriptomic or epigenetic memory from somatic donor cells.
Navarro M. et al.
Embryonic stem cells (ESC) indefinitely maintain the pluripotent state of the blastocyst epiblast. Stem cells are invaluable for studying development and lineage commitment, and in livestock they constitute a useful tool for genomic improvement and in vitro breeding programs. Although these cells have been recently ...

Epigenetic Mechanisms Mediating Cell State Transitions in Chondrocytes
Wuelling M. et al.
Epigenetic modifications play critical roles in regulating cell lineage differentiation, but the epigenetic mechanisms guiding specific differentiation steps within a cell lineage have rarely been investigated. To decipher such mechanisms, we used the defined transition from proliferating (PC) into hypertrophic chon...

Comprehensive characterization of the epigenetic landscape in Multiple Myeloma
Elina Alaterre et al.
Background: Human multiple myeloma (MM) cell lines (HMCLs) have been widely used to understand themolecular processes that drive MM biology. Epigenetic modifications are involved in MM development,progression, and drug resistance. A comprehensive characterization of the epigenetic landscape of MM wouldadvance our un...

Comprehensive characterization of the epigenetic landscape in Multiple Myeloma
Alaterre, Elina and Ovejero, Sara and Herviou, Laurie and de Boussac, Hugues and Papadopoulos, Giorgio and Kulis, Marta and Boireau, Stéphanie and Robert, Nicolas and Requirand, Guilhem and Bruyer, Angélique and Cartron, Guillaume and Vincent, Laure and M
Background: Human multiple myeloma (MM) cell lines (HMCLs) have been widely used to understand the molecular processes that drive MM biology. Epigenetic modifications are involved in MM development, progression, and drug resistance. A comprehensive characterization of the epigenetic landscape of MM would advance our...

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Pettinato, Anthony M. et al.
Human cardiac regeneration is limited by low cardiomyocyte replicative rates and progressive polyploidization by unclear mechanisms. To study this process, we engineer a human cardiomyocyte model to track replication and polyploidization using fluorescently tagged cyclin B1 and cardiac troponin T. Using time-lapse i...

Androgen and glucocorticoid receptor direct distinct transcriptionalprograms by receptor-specific and shared DNA binding sites.
Kulik, Marina et al.
The glucocorticoid (GR) and androgen (AR) receptors execute unique functions in vivo, yet have nearly identical DNA binding specificities. To identify mechanisms that facilitate functional diversification among these transcription factor paralogs, we studied them in an equivalent cellular context. Analysis of chroma...

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Espeso-Gil, S. et al.
In early development, the environment triggers mnemonic epigenomic programs resulting in memory and learning experiences to confer cognitive phenotypes into adulthood. To uncover how environmental stimulation impacts the epigenome and genome organization, we used the paradigm of environmental enrichment (EE) in youn...

TRF2 Mediates Replication Initiation within Human Telomeres to PreventTelomere Dysfunction.
Drosopoulos, William C and Deng, Zhong and Twayana, Shyam and Kosiyatrakul,Settapong T and Vladimirova, Olga and Lieberman, Paul M and Schildkraut,Carl L
The telomeric shelterin protein telomeric repeat-binding factor 2 (TRF2) recruits origin recognition complex (ORC) proteins, the foundational building blocks of DNA replication origins, to telomeres. We seek to determine whether TRF2-recruited ORC proteins give rise to functional origins in telomere repeat tracts. W...

H3K9me3 Antibody (sample size)

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