H3K4me3 Antibody
Polyclonal antibody raised in rabbit against the region of histone H3 containing the trimethylated lysine 4 (H3K4me3), using a KLH-conjugated synthetic peptide.
| 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/ChIP-seq * | 1 µg/ChIP | Fig 1, 2 |
| Dot Blotting | 1:2,000 | Fig 3 |
| Western Blotting | 1:500 | Fig 4 |
| Immunofluorescence | 1:100 | Fig 5 |
* 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
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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.
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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.
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.
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.
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.
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 H3K4 is associated with active promoters.
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) Read more |
| ChIP-seq (ab) Read more |
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 |
Safety sheets
Publications
How to properly cite our product/service in your workWe strongly recommend using this: H3K4me3 Antibody (Hologic Diagenode Cat# C15410030 Lot# 002). Click here to copy to clipboard. Using our products or services in your publication? Let us know! |
SUMO protease FUG1, histone reader AL3 and the PRC1 Complex areintegral to repeat-expansion induced epigenetic silencing in Arabidopsisthaliana |
Temporal modification of H3K9/14ac and H3K4me3 histone marksmediates mechano-responsive gene expression during the accommodationprocess in poplar |
DNA sequence and chromatin modifiers cooperate to confer epigeneticbistability at imprinting control regions. |
Altered Chromatin States Drive Cryptic Transcription in AgingMammalian Stem Cells. |
Age-associated cryptic transcription in mammalian stem cells is linked topermissive chromatin at cryptic promoters |
Identification of Novel Molecular Markers of Human Th17 Cells. |
The Chromatin Factor HNI9 and ELONGATED HYPOCOTYL5 Maintain ROS Homeostasis under High Nitrogen Provision. |
Polycomb Repressive Complex 2 attenuates the very high expression of the Arabidopsis gene NRT2.1. |
Interplay of cell–cell contacts and RhoA/MRTF‐A signaling regulates cardiomyocyte identity |
Neonatal exposure to hyperoxia leads to persistent disturbances in pulmonary histone signatures associated with NOS3 and STAT3 in a mouse model. |
Rapid Communication: The correlation between histone modifications and expression of key genes involved in accumulation of adipose tissue in the pig. |
Lhx2 interacts with the NuRD complex and regulates cortical neuron subtype determinants Fezf2 and Sox11 |
Deep sequencing and de novo assembly of the mouse oocyte transcriptome define the contribution of transcription to the DNA methylation landscape |
CpG signalling, H2A.Z/H3 acetylation and microRNA-mediated deferred self-attenuation orchestrate foetal NOS3 expression. |
Paclitaxel resistance increases oncolytic adenovirus efficacy via upregulated CAR expression and dysfunctional cell cycle control. |
p53-Independent regulation of p21Waf1/Cip1 expression and senescence by PRMT6. |
Transcription and histone methylation changes correlate with imprint acquisition in male germ cells. |