H3K27me3 polyclonal antibody - Classic
(CS-069-100)
As an alternative we offer the purified H3K27me3 polyclonal antibody - Classic.
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| Lot | A299-001 |
|---|---|
| Concentration | not determined |
| Species reactivity | Human |
| Type | Polyclonal |
| Purity | Whole antiserum |
| Host | Rabbit |
| Precautions | This product is for research use only. Not for use in diagnostic or therapeutic procedures. |
| Applications | Suggested dilution | References |
|---|---|---|
| ChIP | 5-10 μl/ ChIP | Fig 1 |
| ELISA | 1:500 | Fig 2 |
| Dot Blotting | 1:20,000 | Fig 3 |
| Western Blotting | 1:1,000 | Fig 4 |
| Immunofluorescence | 1:200 | Fig 5 |
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Validation Data
Figure 1. ChIP results obtained with the Diagenode antibody directed against H3K27me3
ChIP assays were performed using HeLa cells, the Diagenode antibody against H3K27me3 (Cat. No. CS-069-100) and optimized primer sets for qPCR. ChIP was performed with the “OneDay ChIP” kit (Cat. No. kch-oneDIP-060), using sheared chromatin from 1.6 million cells. A titration consisting of 5, 10, and 15 μl of antibody per ChIP experiment was analyzed. IgG (5 μg/IP) was used as a negative IP control. PCR was performed with primers specific for the promoter of the constitutively expressed c-fos gene (Cat. No. pp-1004-050, used as a negative control target) and for the satellite repeat Sat2 (Cat. No. pp-1040-050, used as a positive control target). Figure 1 shows the recovery, expressed as a % of input (the relative amount of immunoprecipitated DNA compared to input DNA after qPCR analysis).
Figure 2. Determination of the antibody titer
To determine the titer of the antibody, an ELISA was performed using a serial dilution of Diagenode antibody directed against H3K27me3 (Cat. No. CS-069-100) in antigen coated wells. The antigen used was a peptide containing the histone modification of interest. By plotting the absorbance against the antibody dilution (Figure 2), the titer of the antibody was estimated to be 1:24,700.
Figure 3. Cross reactivity tests using the Diagenode antibody directed against H3K27me3
A Dot Blot analysis was performed to test the cross reactivity of the Diagenode antibody against H3K27me3 (Cat. No. CS-069-100) with peptides containing other histone modifications including mono- and dimethylation of the same lysine and mono-, di- and trimethylation of other lysines of histone H3. 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 3 shows a high specificity of the antibody for the modification of interest.
Figure 4. Western blot analysis using the Diagenode antibody against H3K27me3
Histone extracts of HeLa cells (HeLa HE, 15 μg) were analysed by Western blot using the Diagenode antibody against H3K27me3 (Cat. No. CS-069-100) 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 Diagenode antibody directed against H3K27me3
NIH3T3 mouse fibroblasts were stained with the Diagenode antibody against H3K27me3 (Cat. No. CS-069-100) and with DAPI. Cells were formaldehyde fixed, permeabilized with Triton X100 and then blocked with PBS containing 1% BSA (Figure 5). (A) Cells were immunofluorescently labelled with the H3K27me3 antibody (diluted 1:200 and incubated for 1 hour at room temperature) followed by goat anti-rabbit antibody conjugated to FITC. (B) Staining of the nuclei with DAPI, which specifically labels DNA. Both antibody and DAPI staining are restricted to the nucleus. H3K27me3 shows a characteristic broadly dispersed pattern in interphase chromatin.
Applications
| ELISA Enzyme-linked immunosorbent assay. Read more |
| 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 |
Documents
| Datasheet H3K27me3 CS-069-100 DATASHEET Datasheet description | Download |
Publications
How to properly cite this product in your workDiagenode strongly recommends using this: H3K27me3 polyclonal antibody - Classic (Diagenode Cat# C15310069 Lot# A299-001 ). Click here to copy to clipboard. Using our products in your publication? Let us know! |
Ordered chromatin changes and human X chromosome reactivation by cell fusion-mediated pluripotent reprogramming |
Spatiotemporal control of estrogen-responsive transcription in ERα-positive breast cancer cells. |
Epstein-Barr virus-mediated transformation of B cells induces global chromatin changes independent to the acquisition of proliferation. |
Genome-wide shRNA screening to identify factors mediating Gata6 repression in mouse embryonic stem cells. |
The histone methyltransferase ASH1 orchestrates fibrogenic gene transcription during myofibroblast transdifferentiation. |
RYBP-PRC1 Complexes Mediate H2A Ubiquitylation at Polycomb Target Sites Independently of PRC2 and H3K27me3. |
Glutathione-S-transferase pi 1(GSTP1) gene silencing in prostate cancer cells is reversed by the histone deacetylase inhibitor depsipeptide. |
Epigenetic silencing mediated through activated PI3K/AKT signaling in breast cancer. |