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 an octameric nucleosome. Histone tails undergo numerous posttranslational 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 are dynamically regulated by histone methyltransferases and histone demethylases, respectively.
H3K4me3 Antibody (sample size)
Monoclonal antibody raised in mouse against histone H3, trimethylated at lysine 4 (H3K4me3), using a KLH-conjugated synthetic peptide.
| Lot | 001-19 |
|---|---|
| Concentration | 1.0 µg/µl |
| Species reactivity | Human, Mouse, Nematodes, Arabidopsis: positive. Other species: not tested. |
| Type | Monoclonal, ChIP-grade, ChIP-seq grade, CUT&Tag grade |
| Purity | Protein A purified |
| Host | Mouse |
| Storage Conditions | Store at -20°C. For long-term storage, store at -80°C. Avoid multiple freeze-thaw cycles. |
| Precautions | This product is for research use only. Not for use in diagnostic or therapeutic procedures. |
| Applications | Suggested dilution | References |
|---|---|---|
| ChIP/ChIP-seq * | 2 μg/ChIP | Fig 1,2 |
| CUT&TAG | 1 μg | Fig 3 |
| ELISA | 1:3,000 | 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 monoclonal antibody directed against H3K4me3
ChIP assays were performed using human HeLa cells, the monoclonal antibody against H3K4me3 (cat. no. C15200152), and optimized PCR primer pairs for qPCR. ChIP was performed with the iDeal ChIP-seq kit (cat. no. C01010051), using sheared chromatin from one million 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 constitutively expressed GAPDH and c-fos genes, used as positive controls, and for exon 2 of the inactive myoglobin (MB) 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 trimethylation of K4 at histone H3 is associated with the promoters of active genes.
Figure 2. ChIP-seq results obtained with the monoclonal antibody directed against H3K4me3
ChIP was performed on sheared chromatin from one million HeLaS3 cells using 2 μg of the antibody against H3K4me3 (cat. no. C15200152) as described above. The IP'd DNA was subsequently analyzed 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 two 5 Mb regions of chromosomes 3 and 5 (Figure 2A and 2B, respectively) and in two 100 kb regions surrounding the GAPDH and c-fos positive control genes (Figure 2C and 2D). These results clearly show an enrichment of H3K4me3 at the promoters of active genes.
Figure 3. CUT&Tag results obtained with the monoclonal antibody directed against H3K4me3
CUT&Tag was performed on 50,000 K562 cells using 1 μg of the monoclonal antibody against H3K4me3 (cat. no. C15200152) and the iDeal CUT&Tag kit (C01070020). The libraries were subsequently analyzed on an Illumina NextSeq 500 sequencer (2 × 75 paired-end reads) according to the manufacturer's instructions. The tags were aligned to the human genome (hg19) using the BWA algorithm. Figure 3 shows the peak distribution along the complete sequence and a 1.5 Mb zoom-in of chromosome 1 (Figure 3A and 3B, respectively).
Figure 4. Cross-reactivity of the monoclonal antibody directed against H3K4me3
To test the specificity, an ELISA was performed using a serial dilution of the monoclonal antibody against H3K4me3 (cat. no. C15200152). The wells were coated with peptides containing the unmodified H3K4 as well as the mono-, di, and trimethylated H3K4 and the trimethylated H3K9. Figure 4 shows a high specificity of the antibody for the modification of interest.
Figure 5. Western blot analysis using the monoclonal antibody directed against H3K4me3
Histone extracts (15 μg) from HeLa cells were analyzed by Western blot using the monoclonal antibody against H3K4me3 (cat. no. C15200152) 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 monoclonal antibody directed against H3K4me3
HeLa cells were stained with the antibody against H3K4me3 (cat. no. C15200152) and with DAPI. Cells were fixed with 4% formaldehyde for 10** min** and blocked with PBS/TX-100 containing 5% normal goat serum and 1% BSA. The cells were immunofluorescently labeled with the H3K4me3 antibody (left) diluted 1:500 in blocking solution, followed by an anti-mouse antibody conjugated to Alexa 594. The middle panel shows staining of the nuclei with DAPI. A merge of the two stains is shown on the right. - Publications
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