| 货号 | 56383S |
| 描述 | SimpleChIP® Plus Sonication Chromatin IP Kit 包含对细胞或组织样品进行多达 24 次染色质免疫沉淀所需的缓冲液和试剂，优化后，每次免疫沉淀可使用 4 X 106 个细胞或 25 mg 组织。一项完整检测可在短达两天内完成，并且可在使用更多或更少细胞或组织样品时随意扩增或缩减。该试剂盒可用于 ChIP-qPCR 和 ChIP-seq。 细胞或组织用甲醛固定后进行裂解，染色质经超声处理被碎裂可获得 200-1000 bp 的染色质片段。基于超声处理的碎裂方法是一种比较传统的染色质碎裂方法，但超声处理应进行优化，才可使用最低的所需超声处理量来获得所需的片段大小，因为过度超声处理会降低免疫沉淀效率，尤其是对于转录因子和辅因子而言。该试剂盒中的细胞与胞核裂解缓冲液经优化可最大化组蛋白、转录因子和辅因子的富集。使用经 ChIP 验证的抗体和 ChIP-Grade Protein G Magnetic Beads 进行染色质免疫沉淀。在蛋白质-DNA 解交联后，DNA 使用 DNA 纯化离心柱进行纯化，以便简单快速地回收 DNA 并去除蛋白污染物，无需进行苯酚/氯仿提取和乙醇沉淀。免疫沉淀期间，特定 DNA 序列的富集可使用标准 PCR、定量实时 PCR、芯片 ChIP 扩增、测序或克隆技术等各种方法进行分析。 SimpleChIP® Plus Kit 还提供确保 ChIP 实验成功的重要对照。该试剂盒包含对人和小鼠核糖体蛋白 L30 (RPL30) 基因进行 PCR 检测所需的阳性对照 Histone H3 Antibody、阴性对照 Normal Rabbit IgG Antibody 和引物组。组蛋白 H3 是染色质的一种核心组分，能在基因组内结合多数 DNA 序列，包括 RPL30 基因座。由于几乎所有被检测基因座上富含Histone H3，因此，Histone H3 Antibody 可用作通用阳性对照。
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| 应用 | CHIP |
| 目标/特异性 | The SimpleChIP® Plus Sonication Chromatin IP Kit can be utilized with ChIP-validated antibodies to detect endogenous levels of protein-DNA interactions and histone modifications in mammalian cells and tissue samples (see Figures 1 to 4). The cell and nuclear lysis buffers for this kit have been optimized to maximize enrichment of chromatin containing histones, transcription factors and cofactors. 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. |
| 供应商 | CST |
| 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).
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| 存放说明 | 4C and -20C |
| 参考文献 | 1 . Orlando, V. (2000) Trends Biochem Sci 25, 99-104. |