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  3. SimpleChIP® Plus Enzymatic Chromatin IP Kit (Agarose Beads)

SimpleChIP® Plus Enzymatic Chromatin IP Kit (Agarose Beads) #9004

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    Product Information

    Storage

    Please store components at the temperatures indicated on the individual tube labels.

    Protocol

    Product Description

    The SimpleChIP® Plus Enzymatic Chromatin IP Kit (Agarose Beads) #9004 contains the buffers and reagents necessary to perform up to 30 chromatin immunoprecipitations from cells or tissue samples, and is optimized for 4 X 106 cells or 25 mg of tissue per immunoprecipitation. A complete assay can be performed in as little as two days and can easily be scaled up or down for use with more or less cells or tissue sample.
    Cells or tissue are fixed with formaldehyde and lysed, and chromatin is fragmented by partial digestion with Micrococcal Nuclease to obtain chromatin fragments of 1 to 5 nucleosomes. Enzymatic fragmentation of chromatin is much milder than sonication and eliminates problems resulting from variability in sonication power and emulsification of chromatin during sonication, which can result in incomplete fragmentation of chromatin or loss of antibody epitopes due to protein denaturation and degradation. Chromatin immunoprecipitations are performed using ChIP-validated antibodies and ChIP-Grade Protein G Agarose Beads. After reversal of protein-DNA cross-links, the DNA is purified using DNA purification spin columns, allowing for easy and efficient recovery of DNA and removal of protein contaminants without the need for phenol/chloroform extractions and ethanol precipitations. The enrichment of particular DNA sequences during immunoprecipitation can be analyzed by a variety of methods, including standard PCR and quantitative real-time PCR. Please note that this kit is not compatible with ChIP-seq because the ChIP-Grade Protein G Agarose Beads are blocked with sonicated salmon sperm DNA, which interferes with downstream sequencing.
    The SimpleChIP® Plus Kit also provides important controls to ensure a successful ChIP experiment. The kit contains a positive control Histone H3 Antibody, a negative control Normal Rabbit IgG Antibody and primer sets for PCR detection of the human and mouse ribosomal protein L30 (RPL30) genes. Histone H3 is a core component of chromatin and is bound to most DNA sequences throughout the genome, including the RPL30 locus. Thus, the Histone H3 Antibody provides a universal positive control that should enrich for almost any locus examined.

    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 and 2). 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.

    Background

    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.

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