H3K4me3 polyclonal antibody

Lot	002
Concentration	1.4 µg/µl
Species reactivity	Human, mouse, Arabidopsis: positive. Other species: not tested.
Type	Polyclonal

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 ^*	1 µg/ChIP	Fig 1, 2
Dot Blotting	1:2,000	Fig 3
Western Blotting	1:500	Fig 4
Immunofluorescence	1:100	Fig 5

A. ChIP

B. ChIP

Figure 1. ChIP results obtained with the Diagenode antibody directed against H3K4me3
ChIP assays were performed using human HeLa cells, the Diagenode antibody against H3K4me3 (Cat. No. C15410030) and optimized PCR primer pairs for qPCR. ChIP was performed with the “iDeal ChIP-seq” kit (cat. No. C01010051), using sheared chromatin from 1 million cells. The chromatin was spiked with a panel of in vitro assembled nucleosomes, each containing a specific lysine methylation (SNAP-ChIP K-MetStat Panel, Epicypher). 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. Figure 1A. Quantitative PCR was performed with primers specific for the promoter of the active GAPDH and EIF4A2 genes, used as positive controls, and for the inactive MYOD1 gene, used as negative control. The graph 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 trimethylation of K4 at histone H3 is associated with the promoters of active genes Figure 1B. Recovery of the nucleosomes carrying the H3K4me1, H3K4me2, H3K4me3 modifications and the unmodified H3K4 as determined by qPCR. The figure clearly shows the antibody is very specific in ChIP for the H3K4me3 modification. At higher concentrations some H3K4me2 is also precipitated.

A. ChIP-seq

B. ChIP-seq

C. ChIP-seq

D. ChIP-seq

Figure 2. ChIP-seq results obtained with the Diagenode antibody directed against H3K4me3
ChIP was performed on sheared chromatin from 1 million HeLa cells using 1 µg of the Diagenode antibody against H3K4me3 (Cat. No. C15410030) as described above. 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 50 bp tags were aligned to the human genome using the BWA algorithm. Figure 2 shows the peak distribution along the complete sequence and a 1.2 Mb region of the X-chromosome (figure 2A and B) and in two regions surrounding the GAPDH and EIF4A2 positive control genes, respectively (figure 2C and D). These results clearly show an enrichment of the H3K4 trimethylation at the promoters of active genes.

Dot Blot

Figure 3. Cross reactivity test using the Diagenode antibody directed against H3K4me3
A Dot Blot analysis was performed to test the cross reactivity of the Diagenode antibody against H3K4me3 (Cat. No. C15410030) with peptides containing other modifications and unmodified sequences of histone H3 and H4. 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 3 shows a high specificity of the antibody for the modification of interest.

Western Blot

Figure 4. Western blot analysis using the Diagenode antibody directed against H3K4me3
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, H3.1, H3.3 and H4 (lane 3, 4, 5, 6 and 7, respectively) using the Diagenode antibody against H3K4me3 (Cat. No. C15410030). The antibody was diluted 1:500 in TBS-Tween containing 5% skimmed milk. The position of the protein of interest is shown on the right, the marker (in kDa) is shown on the left.

Immunofluorescence

Figure 5. Immunofluorescence using the Diagenode antibody directed against H3K4me3
HeLa cells were stained with the Diagenode antibody against H3K4me3 (Cat. No. C15410030) 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 H3K4me3 antibody (left) diluted 1:100 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

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-qPCR (ab)
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ChIP-seq (ab)
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Documents

Datasheet H3K4me3 C15410030 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

Publications

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Interplay of cell–cell contacts and RhoA/MRTF‐A signaling regulates cardiomyocyte identity
Dorn et al
Cell–cell and cell–matrix interactions guide organ development and homeostasis by controlling lineage specification and maintenance, but the underlying molecular principles are largely unknown. Here, we show that in human developing cardiomyocytes cell–cell contacts at the intercalated disk connect...

Rapid Communication: The correlation between histone modifications and expression of key genes involved in accumulation of adipose tissue in the pig.
Kociucka B. et al.
Histone modification is a well-known epigenetic mechanism involved in regulation of gene expression; however, it has been poorly studied in adipose tissues of the pig. Understanding the molecular background of adipose tissue development and function is essential for improving production efficiency and meat quality. ...

Lhx2 interacts with the NuRD complex and regulates cortical neuron subtype determinants Fezf2 and Sox11
Muralidharan B. et al.
n the developing cerebral cortex, sequential transcriptional programs take neuroepithelial cells from proliferating progenitors to differentiated neurons with unique molecular identities. The regulatory changes that occur in the chromatin of the progenitors are not well understood. During deep layer neurogenesis, we...

Deep sequencing and de novo assembly of the mouse oocyte transcriptome define the contribution of transcription to the DNA methylation landscape
Veselovska L, Smallwood SA, Saadeh H, Stewart KR, Krueger F, Maupetit-Méhouas S, Arnaud P, Tomizawa S, Andrews S, Kelsey G
BACKGROUND: Previously, a role was demonstrated for transcription in the acquisition of DNA methylation at imprinted control regions in oocytes. Definition of the oocyte DNA methylome by whole genome approaches revealed that the majority of methylated CpG islands are intragenic and gene bodies are hypermethylated...

CpG signalling, H2A.Z/H3 acetylation and microRNA-mediated deferred self-attenuation orchestrate foetal NOS3 expression.
Postberg J, Kanders M, Forcob S, Willems R, Orth V, Hensel KO, Weil PP, Wirth S, Jenke AC
BACKGROUND: An adverse intrauterine environment leads to permanent physiological changes including vascular tone regulation, potentially influencing the risk for adult vascular diseases. We therefore aimed to monitor responsive NOS3 expression in human umbilical artery endothelial cells (HUAEC) and to study the unde...

Paclitaxel resistance increases oncolytic adenovirus efficacy via upregulated CAR expression and dysfunctional cell cycle control.
Ingemarsdotter CK, Tookman LA, Browne A, Pirlo K, Cutts R, Chelela C, Khurrum KF, Leung EY, Dowson S, Webber L, Khan I, Ennis D, Syed N, Crook TR, Brenton JD, Lockley M, McNeish IA
Resistance to paclitaxel chemotherapy frequently develops in ovarian cancer. Oncolytic adenoviruses are a novel therapy for human malignancies that are being evaluated in early phase trials. However, there are no reliable predictive biomarkers for oncolytic adenovirus activity in ovarian cancer. We investigated the ...

p53-Independent regulation of p21Waf1/Cip1 expression and senescence by PRMT6.
Phalke S, Mzoughi S, Bezzi M, Jennifer N, Mok WC, Low DH, Thike AA, Kuznetsov VA, Tan PH, Voorhoeve PM, Guccione E
p21 is a potent cyclin-dependent kinase inhibitor that plays a role in promoting G1 cell cycle arrest and cellular senescence. Consistent with this role, p21 is a downstream target of several tumour suppressors and oncogenes, and it is downregulated in the majority of tumours, including breast cancer. Here, we repor...

Transcription and histone methylation changes correlate with imprint acquisition in male germ cells.
Henckel A, Chebli K, Kota SK, Arnaud P, Feil R
Genomic imprinting in mammals is controlled by DNA methylation imprints that are acquired in the gametes, at essential sequence elements called 'imprinting control regions' (ICRs). What signals paternal imprint acquisition in male germ cells remains unknown. To address this question, we explored histone methylation ...

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