Diagenode

H3K27me3 polyclonal antibody - Premium (sample size)

Histone-Deacetylase-polyclonal-antibody-diagenode
Catalog Number
Format
Price
C15410195-10
(pAb-195-050)
10 µg
$80.00
  Bulk order
Other format

Polyclonal antibody raised in rabbit against the region of histone H3 containing the trimethylated lysine 27 (H3K27me3), using a KLH-conjugated synthetic peptide.

LotA1811-001P
Concentration1.9 µg/µl
Species reactivityHuman, mouse, C. elegans, Arabidopsis, maize, tomato, poplar, wide range expected
TypePolyclonal
PurityAffinity purified
HostRabbit
PrecautionsThis product is for research use only. Not for use in diagnostic or therapeutic procedures.
Applications Suggested dilution References
ChIP * 1 μg/IP Fig 1, 2
ELISA 1:1,000 Fig 3
Dot Blotting/Peptide array 1:20,000 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 0.5-5 μg per IP.
  • Validation Data

    ChIP

    Figure 1. ChIP results obtained with the Diagenode antibody directed against H3K27me3
    ChIP assays were performed using human K562 cells, the Diagenode antibody against H3K27me3 (cat. No. pAb-195-050) and optimized PCR primer pairs for qPCR. ChIP was performed with the “iDeal ChIP-seq” kit (cat. No. AB-001-0024), using sheared chromatin from 100,000 cells. A titration consisting of 0.1, 0.5, and 1 μg of antibody per ChIP experiment was analyzed. IgG (1 μg/IP) was used as a negative IP control. Quantitative PCR was performed with primers specific for the promoter of the active genes GAPDH and EIF4A2, used as negative controls, and TSH2B and MYT1, used as positive controls. The figure shows the recovery, expressed as a % of input (the relative amount of immunoprecipitated DNA compared to input DNA after qPCR analysis).

    ChIP-seq A ChIP-seq B ChIP-seq C

    Figure 2. ChIP-seq results obtained with the Diagenode antibody directed against H3K27me3
    ChIP was performed on sheared chromatin from 100,000 K562 cells using 1 μg of the Diagenode antibody against H3K27me3 (cat. No. pAb-195-050) 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 36 bp tags were aligned to the human genome using the ELAND algorithm. Figure 2A and B show the signal distribution in two regions surrounding the MYT1 and TSH2B positive control genes, respectively. The position of the PCR amplicon, used for ChIP-qPCR is indicated with an arrow. Figure 2C shows the signal distribution in a 5 Mb region from chromosome 22.

    ELISA

    Figure 3. Determination of the antibody titer
    To determine the titer of the antibody, an ELISA was performed using a serial dilution of the Diagenode antibody against H3K27me3 (cat. No. pAb-195-050). The antigen used was a peptide containing the histone modification of interest. By plotting the absorbance against the antibody dilution (Figure 3), the titer of the antibody was estimated to be 1:22,400.

    Cross reactivity A Cross reactivity B

    Figure 4. Cross reactivity tests using the Diagenode antibody directed against H3K27me3
    Figure 4A. To test the cross reactivity of the Diagenode antibody against H3K27me3 (cat. No. pAb-195-050), a Dot Blot analysis was performed with peptides containing other modifications or 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 1:20,000. Figure 4A shows a high specificity of the antibody for the modification of interest. Figure 4B. The specificity of the antibody was further demonstrated by peptide array analyses on an array containing 384 peptides with different combinations of modifications from histone H3, H4, H2A and H2B. The antibody was used at a dilution of 1:20,000. Figure 4B shows the specificity factor, calculated as the ratio of the average intensity of all spots containing the mark, divided by the average intensity of all spots not containing the mark.

    Western blot

    Figure 5. Western blot analysis using the Diagenode antibody directed against H3K27me3
    Western blot was performed on whole cell (25 μg, lane 1) and histone extracts (15 μg, lane 2) from HeLa cells, and on 1 μg of recombinant histone H2A, H2B, H3 and H4 (lane 3, 4, 5 and 6, respectively) using the Diagenode antibody against H3K27me3 (cat. No. pAb-195-050). The antibody was diluted 1:1,000 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 6. Immunofluorescence using the Diagenode antibody directed against H3K27me3
    Mouse NIH3T3 cells were stained with the Diagenode antibody against H3K27me3 (cat. No. pAb-195-050) 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 labeled with the H3K27me3 antibody (top) diluted 1:500 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 at the bottom.

  • Testimonials

    I am working with the True MicroChIP & Microplex Library Preparation Kits and several histone modification antibodies like H3K27ac, H3K4me3, H3K36me3, and H3K27me3. I got always very good and reproducible results for my ChIP-seq experiments.

    Andrea Thiesen, ZMB, Developmental Biology, Prof. Dr. Andrea Vortkamp´s lab, University Duisburg-Essen, Germany

    In life sciences, epigenetics is nowadays the most rapid developing field with new astonishing discoveries made every day. To keep pace with this field, we are in need of reliable tools to foster our research - tools Diagenode provides us with. From antibodies to automated solutions - all from one source and with robust support. Antibodies used in our lab: H3K27me3 polyclonal antibody – Premium, H3K4me3 polyclonal antibody – Premium, H3K9me3 polyclonal antibody – Premium, H3K4me1 polyclonal antibody – Premium, CTCF polyclonal antibody – Classic, Rabbit IgG.

    Dr. Florian Uhle, Dept. of Anesthesiology, Heidelberg University Hospital, Germany
  • 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
    Peptide array
    Peptide array 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-seq (ab)
    Read more
    ChIP-qPCR (ab)
    Read more
  • Documents
    Datasheet H3K27me3 C15410195 DATASHEET
    Polyclonal antibody raised in rabbit against the region of histone H3 containing the trimethylate...
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    Antibodies you can trust POSTER
    Epigenetic research tools have evolved over time from endpoint PCR to qPCR to the analyses of lar...
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    Epigenetic Antibodies Brochure BROCHURE
    More than in any other immuoprecipitation assays, quality antibodies are critical tools in many e...
    Download
  • Publications

    How to properly cite this product in your work

    Diagenode strongly recommends using this: H3K27me3 polyclonal antibody - Premium (sample size) (Diagenode Cat# C15410195-10 Lot# A1811-001P). Click here to copy to clipboard.

    Using our products in your publication? Let us know!

    Neonatal monocytes exhibit a unique histone modification landscape
    Bermick JR et al.
    Background Neonates have dampened expression of pro-inflammatory cytokines and difficulty clearing pathogens. This makes them uniquely susceptible to infections, but the factors regulating neonatal-specific immune responses are poorly understood. Epigenetics, including histone modifications, can activate or silen...

    BRD4 localization to lineage-specific enhancers is associated with a distinct transcription factor repertoire
    Najafova Z. et al.
    Proper temporal epigenetic regulation of gene expression is essential for cell fate determination and tissue development. The Bromodomain-containing Protein-4 (BRD4) was previously shown to control the transcription of defined subsets of genes in various cell systems. In this study we examined the role of BRD4 in pr...

    Coordinate redeployment of PRC1 proteins suppresses tumor formation during Drosophila development
    Loubiere V. et al.
    Polycomb group proteins form two main complexes, PRC2 and PRC1, which generally coregulate their target genes. Here we show that PRC1 components act as neoplastic tumor suppressors independently of PRC2 function. By mapping the distribution of PRC1 components and trimethylation of histone H3 at Lys27 (H3K27me3) acro...

    Clinical, Imaging, Histopathological and Molecular Characterization of Anaplastic Ganglioglioma
    Zanello M et al.
    Anaplastic ganglioglioma (AGG) is a rare and malignant variant of ganglioglioma. According to the World Health Organization classification version 2016, their histopathological grading criteria are still ill-defined. The aim of the present study was to assess the clinical, imaging, histopathological, and molecular c...

    reChIP-seq reveals widespread bivalency of H3K4me3 and H3K27me3 in CD4(+) memory T cells
    Kinkley S et al.
    The combinatorial action of co-localizing chromatin modifications and regulators determines chromatin structure and function. However, identifying co-localizing chromatin features in a high-throughput manner remains a technical challenge. Here we describe a novel reChIP-seq approach and tailored bioinformatic analys...

    Epigenetic dynamics of monocyte-to-macrophage differentiation
    Wallner S et al.
    BACKGROUND: Monocyte-to-macrophage differentiation involves major biochemical and structural changes. In order to elucidate the role of gene regulatory changes during this process, we used high-throughput sequencing to analyze the complete transcriptome and epigenome of human monocytes that were differentiated in...

    PHF13 is a molecular reader and transcriptional co-regulator of H3K4me2/3
    Chung HR et al.
    PHF13 is a chromatin affiliated protein with a functional role in differentiation, cell division, DNA damage response and higher chromatin order. To gain insight into PHF13's ability to modulate these processes, we elucidate the mechanisms targeting PHF13 to chromatin, its genome wide localization and its molecular ...

    Comprehensive genome and epigenome characterization of CHO cells in response to evolutionary pressures and over time
    Feichtinger J, Hernández I, Fischer C, Hanscho M, Auer N, Hackl M, Jadhav V, Baumann M, Krempl PM, Schmidl C, Farlik M, Schuster M, Merkel A, Sommer A, Heath S, Rico D, Bock C, Thallinger GG, Borth N
    The most striking characteristic of CHO cells is their adaptability, which enables efficient production of proteins as well as growth under a variety of culture conditions, but also results in genomic and phenotypic instability. To investigate the relative contribution of genomic and epigenetic modifications towards...

    Epigenetic regulation of diacylglycerol kinase alpha promotes radiation-induced fibrosis
    Weigel C. et al.
    Radiotherapy is a fundamental part of cancer treatment but its use is limited by the onset of late adverse effects in the normal tissue, especially radiation-induced fibrosis. Since the molecular causes for fibrosis are largely unknown, we analyse if epigenetic regulation might explain inter-individual differences i...

    Standardizing chromatin research: a simple and universal method for ChIP-seq
    Laura Arrigoni, Andreas S. Richter, Emily Betancourt, Kerstin Bruder, Sarah Diehl, Thomas Manke and Ulrike Bönisch
    Here we demonstrate that harmonization of ChIP-seq workflows across cell types and conditions is possible when obtaining chromatin from properly isolated nuclei. We established an ultrasound-based nuclei extraction method (Nuclei Extraction by Sonication) that is highly effective across various organisms, cell ...

    The homeoprotein DLX3 and tumor suppressor p53 co-regulate cell cycle progression and squamous tumor growth
    Palazzo E et al.
    Epidermal homeostasis depends on the coordinated control of keratinocyte cell cycle. Differentiation and the alteration of this balance can result in neoplastic development. Here we report on a novel DLX3-dependent network that constrains epidermal hyperplasia and squamous tumorigenesis. By integrating genetic and t...

    Reinforcement of STAT3 activity reprogrammes human embryonic stem cells to naive-like pluripotency.
    Chen H, Aksoy I, Gonnot F, Osteil P, Aubry M, Hamela C, Rognard C, Hochard A, Voisin S, Fontaine E, Mure M, Afanassieff M, Cleroux E, Guibert S, Chen J, Vallot C, Acloque H, Genthon C, Donnadieu C, De Vos J, Sanlaville D, Guérin JF, Weber M, Stanton LW, R
    Leukemia inhibitory factor (LIF)/STAT3 signalling is a hallmark of naive pluripotency in rodent pluripotent stem cells (PSCs), whereas fibroblast growth factor (FGF)-2 and activin/nodal signalling is required to sustain self-renewal of human PSCs in a condition referred to as the primed state. It is unknown why LIF/...

    A lncRNA regulates alternative splicing via establishment of a splicing-specific chromatin signature.
    Gonzalez I, Munita R, Agirre E, Dittmer TA, Gysling K, Misteli T, Luco RF
    Alternative pre-mRNA splicing is a highly cell type-specific process essential to generating protein diversity. However, the mechanisms responsible for the establishment and maintenance of heritable cell-specific alternative-splicing programs are poorly understood. Recent observations point to a role of histone modi...

    A cohesin-OCT4 complex mediates Sox enhancers to prime an early embryonic lineage.
    Abboud N, Moore-Morris T, Hiriart E, Yang H, Bezerra H, Gualazzi MG, Stefanovic S, Guénantin AC, Evans SM, Pucéat M
    Short- and long-scales intra- and inter-chromosomal interactions are linked to gene transcription, but the molecular events underlying these structures and how they affect cell fate decision during embryonic development are poorly understood. One of the first embryonic cell fate decisions (that is, mesendoderm deter...

    Epigenome mapping reveals distinct modes of gene regulation and widespread enhancer reprogramming by the oncogenic fusion protein EWS-FLI1.
    Tomazou EM, Sheffield NC, Schmidl C, Schuster M, Schönegger A, Datlinger P, Kubicek S, Bock C, Kovar H
    Transcription factor fusion proteins can transform cells by inducing global changes of the transcriptome, often creating a state of oncogene addiction. Here, we investigate the role of epigenetic mechanisms in this process, focusing on Ewing sarcoma cells that are dependent on the EWS-FLI1 fusion protein. We establi...

    Global effects of the CSR-1 RNA interference pathway on the transcriptional landscape.
    Cecere G, Hoersch S, O'Keeffe S, Sachidanandam R, Grishok A
    Argonaute proteins and their small RNA cofactors short interfering RNAs are known to inhibit gene expression at the transcriptional and post-transcriptional levels. In Caenorhabditis elegans, the Argonaute CSR-1 binds thousands of endogenous siRNAs (endo-siRNAs) that are antisense to germline transcripts. However, i...

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