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H3K4me2 Antibody - ChIP-seq Grade

Catalog Number
Format
Price
C15410035
(pAb-035-050)
50 μg
$325.00
  Bulk order
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Polyclonal antibody raised in rabbit against histone H3 containing the dimethylated lysine 4 (H3K4me2), using a KLH-conjugated synthetic peptide.

LotA936-0014P
Concentration1.2 µg/µl
Species reactivityHuman, Arabidopsis: positive. Other species: not tested.
TypePolyclonal
PurityAffinity purified polyclonal antibody.
HostRabbit
Storage ConditionsStore at -20°C; for long storage, store at -80°C. Avoid multiple freeze-thaw cycles.
Storage BufferPBS containing 0.05% azide and 0.05% ProClin 300.
PrecautionsThis product is for research use only. Not for use in diagnostic or therapeutic procedures.
Applications Suggested dilution References
ChIP/ChIP-seq * 0.5 - 1 µg/ChIP Fig 1, 2
ELISA 1:500 Fig 3
Dot Blotting 1:20,000 Fig 4
Western Blotting 1:1,000 Fig 5
Immunofluorescence 1:5,000 Fig 6

* Please note that of the optimal antibody amount per IP should be determined by the end-user. We recommend testing 0.5-5 µg per IP.

  • Validation Data

    H3K4me2 Antibody ChIP Grade

    H3K4me2 Antibody for ChIP

    Figure 1. ChIP results obtained with the Diagenode antibody directed against H3K4me2
    ChIP was performed with the Diagenode antibody against H3K4me2 (Cat. No. C15410035) on sheared chromatin from 500,000 HeLaS3 cells using the “iDeal ChIP-seq” kit (Cat. No. C01010051). 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 of the antibody consisting of 0.5, 1, 2 and 5 µg per ChIP experiment was analysed. IgG (2 µg/IP) was used as negative IP control.
    Figure 1A. Quantitative PCR was performed with primers for a region upstream of the ACTB and GAS2L1 promoters, used as positive controls, and for the MYOD1 gene and the Sat2 satellite repeat, used as negative controls. The graph shows the recovery, expressed as a % of input (the relative amount of immunoprecipitated DNA compared to input DNA after qPCR analysis).
    Figure 1B. Recovery of the nucleosomes carrying the H3K4me1, H3K4me2, H3K4me3, H3K9me2, H3K27me2, H3K36me2, H4K20me2 and the unmodified H3K4 as determined by qPCR. The figure clearly shows the antibody is very specific in ChIP for the H3K4me2 modification.

    A. H3K4me2 Antibody ChIP-seq Grade

    B. H3K4me2 Antibody for ChIP-seq

    C. H3K4me2 Antibody for ChIP-seq assay

    D. H3K4me2 Antibody validated in ChIP-seq

    Figure 2. ChIP-seq results obtained with the Diagenode antibody directed against H3K4me2
    ChIP was performed as described above using 1 µg of the Diagenode antibody against H3K4me2 (Cat. No. C15410035). The IP’d DNA was 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 2 shows the peak distribution along the complete X-chromosome (figure 2A) and in 3 chromosomal regions surrounding the GAPDH, c-fos and ACTB genes (figure 2B, C and D, respectively).

    H3K4me2 Antibody ELISA validation

    Figure 3. Determination of the titer
    To determine the titer, an ELISA was performed using a serial dilution of the Diagenode antibody directed against H3K4me2 (Cat. No. C15410035) 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 3), the titer of the antibody was estimated to be 1:48,200.

    H3K4me2 Antibody validated in Dot Blot

    Figure 4. Cross reactivity test using the Diagenode antibody directed against H3K4me2
    A Dot Blot analysis was performed to test the cross reactivity of the Diagenode antibody against H3K4me2 (Cat. No. C15410035) with peptides containing other modifications of histone H3 and H4 and the unmodified H3K4 sequence. 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 4 shows a high specificity of the antibody for the modification of interest.

    H3K4me2 Antibody validated in Western Blot

    Figure 5. Western blot analysis using the Diagenode antibody directed against H3K4me2
    Histone extracts of HeLa cells (15 µg) were analysed by Western blot using the Diagenode antibody against H3K4me2 (Cat. No. C15410035) 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.

    H3K4me2 Antibody validated in Immunofluorescence

    H3K4me2 Antibody validated in Immunofluorescence

    H3K4me2 Antibody validated in Immunofluorescence

    H3K4me2 Antibody validated for Immunofluorescence

    H3K4me2 Antibody validated for Immunofluorescence

    Figure 6. Immunofluorescence using the Diagenode antibody directed against H3K4me2
    Human osteosarcoma (U2OS) cells were stained with the Diagenode antibody against H3K4me2 (Cat. No. C15410035) and with DAPI. Cells were fixed with 4% formaldehyde for 20’ and blocked with PBS/TX-100 containing 5% normal goat serum. Figure 6A: cells were immunofluorescently labeled with the H3K4me2 antibody (left) diluted 1:5,000 in blocking solution followed by an anti-rabbit antibody conjugated to Alexa568 or with DAPI (right), which specifically labels DNA. Figure 6B, C, D and E: staining of the cells with the H3K4me2 antibody after incubation of the antibody with 5 ng/µl blocking peptide containing the unmodified and the mono-, di- and trimethylated H3K4, respectively.

  • 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
    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-seq (ab)
    Read more
    ChIP-qPCR (ab)
    Read more
  •  Documents
    Datasheet H3K4me2 pAb-035-050 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
  •  Publications

    How to properly cite this product in your work

    Diagenode strongly recommends using this: H3K4me2 Antibody - ChIP-seq Grade (Diagenode Cat# C15410035 Lot# A936-0014P). Click here to copy to clipboard.

    Using our products in your publication? Let us know!

    Inhibition of Histone Demethylases LSD1 and UTX Regulates ERα Signaling in Breast Cancer.
    Benedetti R, Dell'Aversana C, De Marchi T, Rotili D, Liu NQ, Novakovic B, Boccella S, Di Maro S, Cosconati S, Baldi A, Niméus E, Schultz J, Höglund U, Maione S, Papulino C, Chianese U, Iovino F, Federico A, Mai A, Stunnenberg HG, Nebbioso A, Altucci L
    In breast cancer, Lysine-specific demethylase-1 (LSD1) and other lysine demethylases (KDMs), such as Lysine-specific demethylase 6A also known as Ubiquitously transcribed tetratricopeptide repeat, X chromosome (UTX), are co-expressed and co-localize with estrogen receptors (ERs), suggesting the potential use of hybr...

    The Wnt-Driven Mll1 Epigenome Regulates Salivary Gland and Head and Neck Cancer.
    Zhu Q, Fang L, Heuberger J, Kranz A, Schipper J, Scheckenbach K, Vidal RO, Sunaga-Franze DY, Müller M, Wulf-Goldenberg A, Sauer S, Birchmeier W
    We identified a regulatory system that acts downstream of Wnt/β-catenin signaling in salivary gland and head and neck carcinomas. We show in a mouse tumor model of K14-Cre-induced Wnt/β-catenin gain-of-function and Bmpr1a loss-of-function mutations that tumor-propagating cells exhibit increased Mll1 activi...

    Histone variant H2A.Z deposition and acetylation directs the canonical Notch signaling response.
    Giaimo BD, Ferrante F, Vallejo DM, Hein K, Gutierrez-Perez I, Nist A, Stiewe T, Mittler G, Herold S, Zimmermann T, Bartkuhn M, Schwarz P, Oswald F, Dominguez M, Borggrefe T
    A fundamental as yet incompletely understood feature of Notch signal transduction is a transcriptional shift from repression to activation that depends on chromatin regulation mediated by transcription factor RBP-J and associated cofactors. Incorporation of histone variants alter the functional properties of chromat...

    The Arabidopsis LDL1/2-HDA6 histone modification complex is functionally associated with CCA1/LHY in regulation of circadian clock genes.
    Hung FY, Chen FF, Li C, Chen C, Lai YC, Chen JH, Cui Y, Wu K
    In Arabidopsis, the circadian clock central oscillator genes are important cellular components to generate and maintain circadian rhythms. There is a negative feedback loop between the morning expressed CCA1 (CIRCADIAN CLOCK ASSOCIATED 1)/LHY (LATE ELONGATED HYPOCOTYL) and evening expressed TOC1 (TIMING OF CAB EXPRE...

    SETBP1 induces transcription of a network of development genes by acting as an epigenetic hub.
    Piazza R, Magistroni V, Redaelli S, Mauri M, Massimino L, Sessa A, Peronaci M, Lalowski M, Soliymani R, Mezzatesta C, Pirola A, Banfi F, Rubio A, Rea D, Stagno F, Usala E, Martino B, Campiotti L, Merli M, Passamonti F, Onida F, Morotti A, Pavesi F, Bregni
    SETBP1 variants occur as somatic mutations in several hematological malignancies such as atypical chronic myeloid leukemia and as de novo germline mutations in the Schinzel-Giedion syndrome. Here we show that SETBP1 binds to gDNA in AT-rich promoter regions, causing activation of gene expression through recruitment ...

    A CLK3-HMGA2 Alternative Splicing Axis Impacts Human Hematopoietic Stem Cell Molecular Identity throughout Development.
    Cesana M, Guo MH, Cacchiarelli D, Wahlster L, Barragan J, Doulatov S, Vo LT, Salvatori B, Trapnell C, Clement K, Cahan P, Tsanov KM, Sousa PM, Tazon-Vega B, Bolondi A, Giorgi FM, Califano A, Rinn JL, Meissner A, Hirschhorn JN, Daley GQ
    While gene expression dynamics have been extensively cataloged during hematopoietic differentiation in the adult, less is known about transcriptome diversity of human hematopoietic stem cells (HSCs) during development. To characterize transcriptional and post-transcriptional changes in HSCs during development, we le...

    Overexpression of histone demethylase Fbxl10 leads to enhanced migration in mouse embryonic fibroblasts.
    Rohde M. et al.
    Cell migration is a central process in the development and maintenance of multicellular organisms. Tissue formation during embryonic development, wound healing, immune responses and invasive tumors all require the orchestrated movement of cells to specific locations. Histone demethylase proteins alter transcription ...

    A novel microscopy-based high-throughput screening method to identify proteins that regulate global histone modification levels.
    Baas R, Lelieveld D, van Teeffelen H, Lijnzaad P, Castelijns B, van Schaik FM, Vermeulen M, Egan DA, Timmers HT, de Graaf P
    Posttranslational modifications of histones play an important role in the regulation of gene expression and chromatin structure in eukaryotes. The balance between chromatin factors depositing (writers) and removing (erasers) histone marks regulates the steady-state levels of chromatin modifications. Here we describe...

    The H3K4me3 histone demethylase Fbxl10 is a regulator of chemokine expression, cellular morphology and the metabolome of fibroblasts
    Janzer A, Stamm K, Becker A, Zimmer A, Buettner R, Kirfel J
    Fbxl10 (Jhdm1b/Kdm2b) is a conserved and ubiquitously expressed member of the JHDM (JmjC-domain-containing histone demethy-lase) family. Fbxl10 was implicated in the demethylation of H3K4me3 or H3K36me2 thereby removing active chromatin marks and inhibiting gene transcription. Apart from the JmjC domain, Fbxl10 cons...

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