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  • Chromatin remodeling gene AT-rich interactive domain-containing protein 1A suppresses gastric cancer cell proliferation by targeting PIK3CA and PDK1. 27323812

    The tumor suppressor gene AT-rich interactive domain-containing protein 1A (ARID1A) was frequently mutated in cancers. The modulation mechanism of ARID1A for PI3K/AKT signaling in gastric cancer (GC) remains elusive. Here, we found that depletion of endogenous ARID1A enhanced the in vitro proliferation, colony formation, cellular growth, nutrient uptake and in vivo xenograft tumor growth of GC cells. PI3K/AKT activation by ARID1A-silencing was profiled using a phospho-protein antibody array. The phosphorylation of PDK1, AKT, GSK3β and 70S6K, and the protein and mRNA expressions of PI3K and PDK1, were upregulated by ARID1A-silencing. Chromatin immunoprecipitation and luciferase reporter assay revealed that ARID1A-involved SWI/SNF complex inhibited PIK3CA and PDK1 transcription by direct binding to their promoters. Serial deletion mutation analyses revealed that the ARID1A central region containing the HIC1-binding domain, but not the ARID DNA-binding domain and the C-terminal domain, was essential for the inhibition of GC cell growth, PI3K/AKT pathway phosphorylation and its transcriptional modulation activity of PIK3CA and PDK1. The proliferation, cellular growth and glucose consumption of ARID1A-deficient GC cells were efficiently prohibited by allosteric inhibitors mk2206 and LY294002, which targeting AKT and PI3K, respectively. Both inhibitors also downregulated the phosphorylation of PI3K/AKT pathway in ARID1A-deficient GC cells. Such cells were sensitized to the treatment of LY294002, and AT7867, another inhibitor of AKT and p70S6K. The administration of LY294002 alone inhibited the in vivo growth of ARID1A- deficient GC cells in mouse xenograft model. Our study provides a novel insight into the modulatory function and mechanism of ARID1A in PI3K/AKT signaling in GC.
    Dokumententyp:
    Referenz
    Produkbestellnummer:
    17-371
    Produktbezeichnung:
    EZ-ChIP™

  • Insertional chromatin immunoprecipitation: a method for isolating specific genomic regions. 19804873

    We established a novel method, insertional chromatin immunoprecipitation (iChIP), for isolation of specific genomic regions. In iChIP, specific genomic domains are immunoprecipitated with antibody against a tag, which is fused to the DNA-binding domain of an exogenous DNA-binding protein, whose recognition sequence is inserted into the genomic domains of interest. The iChIP method will be a useful tool for dissecting chromatin structure of genomic region of interest.
    Dokumententyp:
    Referenz
    Produkbestellnummer:
    Mehrere
    Produktbezeichnung:
    Mehrere

  • Megabase chromatin domains involved in DNA double-strand breaks in vivo. 10477747

    The loss of chromosomal integrity from DNA double-strand breaks introduced into mammalian cells by ionizing radiation results in the specific phosphorylation of histone H2AX on serine residue 139, yielding a specific modified form named gamma-H2AX. An antibody prepared to the unique region of human gamma-H2AX shows that H2AX homologues are phosphorylated not only in irradiated mammalian cells but also in irradiated cells from other species, including Xenopus laevis, Drosophila melanogaster, and Saccharomyces cerevisiae. The antibody reveals that gamma-H2AX appears as discrete nuclear foci within 1 min after exposure of cells to ionizing radiation. The numbers of these foci are comparable to the numbers of induced DNA double-strand breaks. When DNA double-strand breaks are introduced into specific partial nuclear volumes of cells by means of a pulsed microbeam laser, gamma-H2AX foci form at these sites. In mitotic cells from cultures exposed to nonlethal amounts of ionizing radiation, gamma-H2AX foci form band-like structures on chromosome arms and on the end of broken arms. These results offer direct visual confirmation that gamma-H2AX forms en masse at chromosomal sites of DNA double-strand breaks. The results further suggest the possible existence of units of higher order chromatin structure involved in monitoring DNA integrity.
    Dokumententyp:
    Referenz
    Produkbestellnummer:
    07-164
    Produktbezeichnung:
    Anti-phospho-H2A.X (Ser139) Antibody

  • A fast carrier chromatin immunoprecipitation method applicable to microdissected tissue samples. 18502516

    Transcriptional regulation studies of CNS neurons are complicated by both cellular diversity and plasticity. Microdissection of specific functionally related populations of neurons can greatly reduce these issues, but typically excludes the use of many technologies due to tissue requirements, such as Chromatin Immunoprecipitation (ChIP), a powerful tool for studying in vivo protein-DNA interactions. We have developed a fast carrier ChIP (Fast CChIP) method for analyzing specific in vivo transcription factor-DNA interactions in as little as 0.2 mm(3) brain tissue. Using an antibody against phosphorylated cyclic-AMP response element binding (CREB) protein, we confirmed phospho-CREB (pCREB) binding at the c-fos gene promoter. Then we further demonstrated the applicability of Fast CChIP in determining hypertension-induced pCREB binding at the c-fos gene promoter in the rat nucleus tractus solitarius (NTS), confirming CREB's role in mediating hypertension-induced c-fos expression. This method will be broadly applicable to individual brain nucleus and biopsy/surgical samples.
    Dokumententyp:
    Referenz
    Produkbestellnummer:
    Mehrere
    Produktbezeichnung:
    Mehrere

  • Q2ChIP, a quick and quantitative chromatin immunoprecipitation assay, unravels epigenetic dynamics of developmentally regulated genes in human carcinoma cells. 17272500

    Chromatin immunoprecipitation (ChIP) is a key technique for studying protein-DNA interactions and mapping epigenetic histone modifications on DNA. Current ChIP protocols require extensive sample handling and large cell numbers. We developed a quick and quantitative (Q(2))ChIP assay suitable for histone and transcription factor immunoprecipitation from chromatin amounts equivalent to as few as 100 cells. DNA-protein cross-linking in suspension in presence of butyrate, elimination of background chromatin through a tube shift after washes, and a combination of cross-link reversal, protein digestion, increased antibody-bead to chromatin ratio, and DNA elution into a single step considerably improve ChIP efficiency and shorten the procedure. We used Q(2)ChIP to monitor changes in histone H3 modifications on the 5' regulatory regions of the developmentally regulated genes OCT4, NANOG, LMNA, and PAX6 in the context of retinoic-acid-mediated human embryonal carcinoma cell differentiation. Quantitative polymerase chain reaction analysis of precipitated DNA unravels biphasic heterochromatin assembly on OCT4 and NANOG, involving H3 lysine (K)9 and K27 methylation followed by H3K9 deacetylation and additional H3K27 trimethylation. Di- and trimethylation of H3K4 remain relatively unaltered. In contrast, PAX6 displays histone modifications characteristic of repressed genes with potential for activation in undifferentiated cells. PAX6 undergoes H3K9 acetylation and enhanced H3K4 trimethylation upon transcriptional activation. Q(2)ChIP of the transcription factor Oct4 demonstrates its dissociation from the NANOG promoter upon differentiation. This study is, to our knowledge, the first to reveal histone modification changes on human OCT4 and NANOG regulatory sequences. The results demonstrate ordered chromatin rearrangement on developmentally regulated promoters upon differentiation.
    Dokumententyp:
    Referenz
    Produkbestellnummer:
    07-442
    Produktbezeichnung:
    Anti-trimethyl-Histone H3 (Lys9) Antibody

  • Protocol: methodology for chromatin immunoprecipitation (ChIP) in Chlamydomonas reinhardtii. 22050920

    We report on a detailed chromatin immunoprecipitation (ChIP) protocol for the unicellular green alga Chlamydomonas reinhardtii. The protocol is suitable for the analysis of nucleosome occupancy, histone modifications and transcription factor binding sites at the level of mononucleosomes for targeted and genome-wide studies. We describe the optimization of conditions for crosslinking, chromatin fragmentation and antibody titer determination and provide recommendations and an example for the normalization of ChIP results as determined by real-time PCR.
    Dokumententyp:
    Referenz
    Produkbestellnummer:
    Mehrere
    Produktbezeichnung:
    Mehrere

  • Nuclear IGF-1R interacts with regulatory regions of chromatin to promote RNA polymerase II recruitment and gene expression associated with advanced tumor stage. 29735545

    Internalization of ligand-activated type 1 IGF receptor (IGF-1R) is followed by recycling to the plasma membrane, degradation or nuclear translocation. Nuclear IGF-1R reportedly associates with clinical response to IGF-1R inhibitory drugs, yet its role in the nucleus is poorly characterized. Here we investigated the significance of nuclear IGF-1R in clinical cancers and cell line models. In prostate cancers, IGF-1R was predominantly membrane-localized in benign glands, while malignant epithelium contained prominent internalized (nuclear/cytoplasmic) IGF-1R, and nuclear IGF-1R associated significantly with advanced tumor stage. Using ChIP-seq to assess global chromatin occupancy, we identified IGF-1R binding sites at or near transcription start sites of genes including JUN and FAM21, most sites coinciding with occupancy by RNA polymerase II (RNAPol2) and histone marks of active enhancers/promoters. IGF-1R was inducibly recruited to chromatin, directly binding DNA and interacting with RNAPol2 to upregulate expression of JUN and FAM21, shown to mediate tumor cell survival and IGF-induced migration. IGF-1 also enriched RNAPol2 on promoters containing IGF-1R binding sites. These functions were inhibited by IGF-1/2 neutralizing antibody xentuzumab (BI 836845), or by blocking receptor internalization. We detected nuclear IGF-1R on JUN and FAM21 promoters in fresh prostate cancers that contained abundant nuclear IGF-1R, with evidence of correlation between nuclear IGF-1R content and JUN expression in malignant prostatic epithelium. Taken together, these data reveal previously unrecognized molecular mechanisms through which IGFs promote tumorigenesis, with implications for therapeutic evaluation of anti-IGF drugs.
    Dokumententyp:
    Referenz
    Produkbestellnummer:
    17-295
    Produktbezeichnung:
    Chromatin Immunoprecipitation (ChIP) Assay Kit

  • High resolution analysis of the chromatin landscape of the IgE switch region in human B cells. 21949728

    Antibodies are assembled by a highly orchestrated series of recombination events during B cell development. One of these events, class switch recombination, is required to produce the IgG, IgE and IgA antibody isotypes characteristic of a secondary immune response. The action of the enzyme activation induced cytidine deaminase is now known to be essential for the initiation of this recombination event. Previous studies have demonstrated that the immunoglobulin switch regions acquire distinct histone modifications prior to recombination. We now present a high resolution analysis of these histone modifications across the IgE switch region prior to the initiation of class switch recombination in primary human B cells and the human CL-01 B cell line. These data show that upon stimulation with IL-4 and an anti-CD40 antibody that mimics T cell help, the nucleosomes of the switch regions are highly modified on histone H3, accumulating acetylation marks and tri-methylation of lysine 4. Distinct peaks of modified histones are found across the switch region, most notably at the 5' splice donor site of the germline (I) exon, which also accumulates AID. These data suggest that acetylation and K4 tri-methylation of histone H3 may represent marks of recombinationally active chromatin and further implicates splicing in the regulation of AID action.
    Dokumententyp:
    Referenz
    Produkbestellnummer:
    Mehrere
    Produktbezeichnung:
    Mehrere

  • Regulation of extracellular chromatin release from neutrophils. 20375577

    Neutrophils use intricate mechanisms for capturing and killing invading microorganisms. One mechanism entails the release of relaxed chromatin from the cell. Microbes are trapped by the extracellular chromatin and exposed to high local concentrations of bactericidal compounds. We examine the regulation of chromatin release by testing the contribution of microtubules and the actin cytoskeleton to the deployment of neutrophil extracellular traps (NETs). Incubation of human neutrophils with nocodazole, a tubulin polymerization inhibitor, or cytochalasin D, an inhibitor of actin filamentation, severely diminished the ability of neutrophils to respond to LPS by releasing chromatin from the cells. In addition, pretreatment of neutrophils with M1/70, a monoclonal antibody to the Mac-1 integrin adhesion receptor, drastically reduced the deployment of chromatin into NETs. Analysis of histone deimination, the conversion of arginine to citrulline in 3 of the 4 core histones by peptidylarginine deiminase 4, revealed that the treatments inhibiting NET formation also reduced histone deimination. Our data indicate that NET formation requires functional tubulin and actin filaments and responds to engagement of Mac-1 integrins. Because histone deimination coincides with the release of NETs, we propose that these events represent overlapping mechanisms of neutrophil responses to infections.
    Dokumententyp:
    Referenz
    Produkbestellnummer:
    07-690
    Produktbezeichnung:
    Anti-Histone H3 Antibody, CT, pan

  • The D-isoAsp-25 variant of histone H2B is highly enriched in active chromatin: potential role in the regulation of gene expression? 26666674

    Approximately 12 % of histone H2B in mammalian brain contains an unusual D-aspartate residue in its N-terminal tail. Most of this D-aspartate is linked to the C-flanking glycine via an isopeptide bond. To explore the possible significance of these modifications, we generated an antibody to the D-isoaspartyl form of H2B, and used it to assess its levels in H2B associated with "active" vs. "silent" chromatin. We found that the D-isoaspartyl form of H2B appears to be highly enriched in the former. This irreversible modification could serve a novel regulatory function in gene expression.
    Dokumententyp:
    Referenz
    Produkbestellnummer:
    17-10086
    Produktbezeichnung:
    EZ-Magna ChIP™ A/G Chromatin Immunoprecipitation Kit