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SimpleChIP ® Plus Enzymatic Chromatin IP Kit (Magnetic Beads)
SimpleChIP ® Plus Enzymatic Chromatin IP Kit (Magnetic Beads)
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SimpleChIP ® Plus Enzymatic Chromatin IP Kit (Magnetic Beads)

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    实验应用：
    ChIP
    ChIP
    产品介绍
    产品信息
    产品详情
    Chromatin Regulation / Nuclear Function
    简单描述
    SimpleChIP® Plus enzymatic ChIP kit for use with both cells and tissues. Highly amenable to the study of transcription factors and cofactors.
    组合货号
    38191S&66816S
    组成成分
    蛋白酶K，IgG 抗体，核酸外切酶，丙三醇 (甘油)，迭氮(化)钠,水
    应用
    实验应用
    ChIP
    目标/特异性

    Specificity/Sensitivity

    The SimpleChIP® Plus Enzymatic Chromatin IP Kit can be utilized with any ChIP-validated antibody to detect endogenous levels of protein-DNA interactions and histone modifications in mammalian cells and tissue samples (see Figures 1-6). The positive control Histone H3 Antibody recognizes many different species of the highly conserved Histone H3 protein, including human, mouse, rat and monkey. Primer sets are included for the human and mouse positive control RPL30 gene loci; however, the use of other species with the kit requires the design of additional control primer sets.

    背景
    背景
    The chromatin immunoprecipitation (ChIP) assay is a powerful and versatile technique used for probing protein-DNA interactions within the natural chromatin context of the cell (1,2). This assay can be used to identify multiple proteins associated with a specific region of the genome, or the opposite, to identify the many regions of the genome bound by a particular protein (3-6). It can be used to determine the specific order of recruitment of various proteins to a gene promoter or to "measure" the relative amount of a particular histone modification across an entire gene locus (3,4). In addition to histone proteins, the ChIP assay can be used to analyze binding of transcription factors and co-factors, DNA replication factors and DNA repair proteins. When performing the ChIP assay, cells or tissues are first fixed with formaldehyde, a reversible protein-DNA cross-linking agent that "preserves" the protein-DNA interactions occurring in the cell (1,2). Cells are lysed and chromatin is harvested and fragmented using either sonication or enzymatic digestion. The chromatin is then immunoprecipitated with antibodies specific to a particular protein or histone modification. Any DNA sequences that are associated with the protein or histone modification of interest will co-precipitate as part of the cross-linked chromatin complex and the relative amount of that DNA sequence will be enriched by the immunoselection process. After immunoprecipitation, the protein-DNA cross-links are reversed and the DNA is purified. Standard PCR or Quantitative Real-Time PCR can be used to measure the amount of enrichment of a particular DNA sequence by a protein-specific immunoprecipitation (1,2). Alternatively, the ChIP assay can be combined with genomic tiling micro-array (ChIP on chip) techniques, high throughput sequencing, or cloning strategies, all of which allow for genome-wide analysis of protein-DNA interactions and histone modifications (5-8). 1.Orlando, V. (2000) Trends Biochem Sci 25, 99-104. 2.Liu, Q. et al. (2000) Genes Dev 14, 1448-59. 3.Kuo, M.H. and Allis, C.D. (1999) Methods 19, 425-33. 4.Zhao, H. and Piwnica-Worms, H. (2001) Mol Cell Biol 21, 4129-39. 5.Agalioti, T. et al. (2000) Cell 103, 667-78. 6.Jiang, K. et al. (2003) J Biol Chem 278, 25207-17. 7.Soutoglou, E. and Talianidis, I. (2002) Science 295, 1901-4. 8.Martin, S.A. and Ouchi, T. (2008) Mol Cancer Ther 7, 2509-16. 9.Mikkelsen, T.S. et al. (2007) Nature 448, 553-60. 10.Chen, M.S. et al. (2003) Mol Cell Biol 23, 7488-97. 11.Lee, T.I. et al. (2006) Cell 125, 301-13. 12.Zeng, Y. et al. (1998) Nature 395, 507-10. 13.Weinmann, A.S. and Farnham, P.J. (2002) Methods 26, 37-47. 14.Löffler, H. et al. (2006) Cell Cycle 5, 2543-7. 15.Wells, J. and Farnham, P.J. (2002) Methods 26, 48-56. 16.Zachos, G. et al. (2007) Dev Cell 12, 247-60. 17.Garber, K. (2005) J Natl Cancer Inst 97, 1026-8.
    制备和贮存
    保存方式
    Please store components at the temperatures indicated on the individual tube labels.

    参考图片

    FIGURE 3. Mouse brain and mouse liver tissue were prepared and disaggregated into a single-cell suspension using a Dounce homogenizer and tissue disaggregator, respectively. The cells were then formaldehyde-crosslinked and chromatin was prepared and digested. DNA was purified and 10 μl was separated by electrophoresis on a 1% agarose gel and stained with ethidium bromide. The majority of chromatin from both brain (lane 1) and liver (lane 2) was digested to 1 to 5 nucleosomes in length (150 to 900 bp).

    图3：使用Dounce高速搅拌器和组织分离器，小鼠大脑和小鼠肝脏组织分别被准备以及被分离成一个单个细胞悬液。细胞被甲醛固定-交联以及染色质被准备和消化。DNA被纯化以及通过1%琼脂糖凝胶电泳分离10 μl和使用溴化乙锭染色。绝大部分大脑(第1道)和肝脏(第2道)染色质被消化成1到5核小体，在长度上(150 到 900 bp)。

    FIGURE 1. Mouse brain was prepared and disaggregated into a single-cell suspension using a Dounce homogenizer. The cells were then cross-linked with formaldehyde, and chromatin was prepared and digested. Chromatin immunoprecipitations were performed using the indicated ChIP-validated antibodies. Purified DNA was analyzed by quantitative real-time PCR using SimpleChIP® Mouse GAPDH Intron 2 Primers #8986, SimpleChIP® Mouse RPL30 Intron 2 Primers #7015, SimpleChIP® Mouse HoxA1 Promoter Primers #7341, and SimpleChIP® Mouse HoxD10 Exon 1 Primers #7429. The amount of immunoprecipitated DNA in each sample is represented as signal relative to the total amount of input chromatin (equivalent to 1).

    图1：使用Dounce高速搅拌器，小鼠大脑被准备，并且被分离成一个单个细胞悬液。细胞与甲醛交联，以及染色质被准备和消化。使用已制定的ChIP-validated antibodies进行免疫沉淀实验。使用SimpleChIP® Mouse GAPDH Intron 2 Primers #8986、SimpleChIP® Mouse RPL30 Intron 2 Primers #7015,、SimpleChIP® Mouse HoxA1 Promoter Primers #7341和SimpleChIP® Mouse HoxD10 Exon 1 Primers #7429，通过quantitative real-time PCR分析纯化的DNA。在每个样品中免疫沉淀DNA的量被看作相对于input chromatin总量 (相当于1)的信号。

    FIGURE 2. Mouse liver was prepared and disaggregated into a single-cell suspension using a tissue disaggregator. The cells were then cross-linked with formaldehyde, and chromatin was prepared and digested. Chromatin immunoprecipitations were performed using the indicated ChIP-validated antibodies. Purified DNA was analyzed by quantitative real-time PCR using SimpleChIP® Mouse GAPDH Intron 2 Primers #8986, SimpleChIP® Mouse AFM Intron 2 Primers #7269, SimpleChIP® Mouse HoxA1 Promoter Primers #7341, and SimpleChIP® Mouse HoxD10 Exon 1 Primers #7429. The amount of immunoprecipitated DNA in each sample is represented as signal relative to the total amount of input chromatin (equivalent to 1).

    图2：使用组织分离器，小鼠肝脏被准备，并且被分离成一个单个细胞悬液。细胞与甲醛交联，以及染色质被准备和消化。使用已制定的ChIP-validated antibodies进行免疫沉淀实验。使用SimpleChIP® Mouse GAPDH Intron 2 Primers #8986、SimpleChIP® Mouse AFM Intron 2 Primers #7269、SimpleChIP® Mouse HoxA1 Promoter Primers #7341和SimpleChIP® Mouse HoxD10 Exon 1 Primers #7429，通过quantitative real-time PCR分析纯化的DNA。在每个样品中免疫沉淀DNA的量被看作相对于input chromatin总量 (相当于1)的信号。

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    货号：
    9005S
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