H3K27me3 Antibody
Monoclonal antibody raised in mouse against histone H3 trimethylated at lysine 27 (H3K27me3), using a KLH-conjugated synthetic peptide.
| Lot | 001-16 |
|---|---|
| Concentration | 1 µg/µl |
| Species reactivity | Human, Nematodes, Magnaporthe oryzae: positive. Other species: not tested. |
| Type | Monoclonal |
| Purity | Protein A purified monoclonal antibody. |
| Host | Mouse |
| 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. |
| Precautions | This product is for research use only. Not for use in diagnostic or therapeutic procedures. |
| Applications | Suggested dilution | References |
|---|---|---|
| ChIP * | 1-2 µg/ChIP | Fig 1 |
| CUT&TAG | 1 µg | Fig 2 |
| ELISA | 1:3,000 | Fig 3 |
| Western Blotting | 1:1,000 | Fig 4 |
| Immunofluorescence | 1:500 | 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
Figure 1. ChIP results obtained with the monoclonal antibody directed against H3K27me3
ChIP assays were performed using human HeLa cells, the monoclonal antibody against H3K27me3 (cat. No. C15200181) and optimized PCR primer sets for qPCR. ChIP was performed with the “iDeal ChIP-seq” kit (cat. No. C01010051) on sheared chromatin from 1 million cells. A titration of the antibody consisting of 1, 2, 5, and 10 µg per ChIP experiment was analysed. IgG (2 µg/IP) was used as negative IP control. QPCR was performed with primers for the promoters of the active genes c-fos (cat. No. C17011004) and GAPDH as negative controls, and for the coding regions of the inactive genes MYT1 and TSH2B (cat. No. C17011041) as positive 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 H3K27me3 is preferably present at inactive genes.
Figure 2. Cut&Tag results obtained with the monoclonal antibody directed against H3K27me3
CUT&TAG (Kaya-Okur, H.S., Nat Commun 10, 1930, 2019) was performed on 50,000 K562 cells using 1 µg of the monoclonal antibody against H3K27me3 (cat. No. C15200181) and the Diagenode pA-Tn5 transposase (C01070001). The libraries were subsequently analysed on an Illumina NextSeq 500 sequencer (2x75 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 in 2 genomic regions on chromosome 3 and 20 (figure 2A and B, respectively).
Figure 3. Cross reactivity of the monoclonal antibody directed against H3K27me3
To test the specificity an ELISA was performed using a serial dilution of the monoclonal antibody against H3K27me3 (cat. No. C15200181). The wells were coated with peptides containing the unmodified H3K27 region as well as the mono-, di- and trimethylated H3K27 and the trimethylated H3K9. Figure 3 shows a high specificity of the antibody for the peptide containing the modification of interest.
Figure 4. Western blot analysis using the monoclonal antibody directed against H3K27me3
Histone extracts (15 µg) from HeLa cells were analysed by Western blot using the monoclonal antibody against H3K27me3 (cat. No. C15200181) 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 5. Immunofluorescence using the monoclonal antibody directed against H3K27me3
HeLa cells were stained with the antibody against H3K27me3 (cat. No. C15200181) 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 H3K27me3 antibody (left) diluted 1:500 in blocking solution followed by an anti-mouse antibody conjugated to Alexa594. 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.
Applications
| ELISA Enzyme-linked immunosorbent assay. 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 |
| 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 H3K27me3 C15200181 DATASHEET Datasheet description | Download |
| Epigenetic Antibodies Brochure BROCHURE More than in any other immuoprecipitation assays, quality antibodies are critical tools in many e... | Download |
| Antibodies you can trust POSTER Epigenetic research tools have evolved over time from endpoint PCR to qPCR to the analyses of lar... | Download |
Safety sheets
Publications
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