CST的SimpleChIP® 酶解染色质免疫沉淀试剂盒是由CST与New England Biolabs的科学家联合开发的，因此包含了最高质量的研究试剂。该试剂盒有两种：Protein G 琼脂糖微球和Protein G 磁珠，所包含的缓冲液和试剂足够做30次ChIP实验。Protein G 琼脂微球和Protein G 磁珠也有单独的产品。

酶消化染色质比超声破碎更加温和。温和的处理方式更好保留了染色质完整性和蛋白的抗原表位， 增加了免疫沉淀的效率。

详细的操作步骤专为细胞和组织样品优化，节约您的时间和试剂。

试剂盒可与任何ChIP级抗体一起检测哺乳动物细胞内源水平的蛋白质-DNA相互作用和组蛋白修饰。

包含所有试剂和对照，节约时间，提供合适的实验对照。

试剂盒内的试剂在公司内部用于验证我们的ChIP 抗体，将方便您的优化。

试剂盒内包含的ChIP 级磁珠非常适于下游的ChIP-on-chip 或 ChIP 测序，因为其更高的灵敏度和低背景。

9002       SimpleChIP® Enzymatic Chromatin IP Kit (Agarose Beads)    

9003       SimpleChIP® Enzymatic Chromatin IP Kit (Magnetic Beads)

SimpleChIP® Enzymatic Chromatin IP Kit (Agarose Beads)包含缓冲液和试剂，必须进行6次染色质准备和30次染色质免疫沉淀，并且每次实验最佳的细胞数为4 X 107 。一个完整的实验至少两天时间被完成，并且对于使用多的或少的细胞数试剂盒很容易扩大或缩小。细胞使用甲醛固定，并且通过微球菌核酸酶部分程度上酶切染色质成片段，从而获得1-5个核小体的染色质片段。染色质的酶切片段是比超声更加温和以及消除了由超声功率的可比性以及在超声讲解中染色质的乳化作用带来的问题，这能够导致染色质的不完全片段化或由于蛋白质变性和降解造成的抗体表位的损失。使用ChIP-validated antibodies和ChIP-Grade Protein G Agarose Beads进行染色质免疫沉淀。在蛋白质-DNA交联的逆转之后，使用DNA纯化旋转柱被纯化，这允许DNA更容易和高效的恢复，并且蛋白质的移除不需要苯酚/氯仿的提取和乙醇的沉淀。在免疫沉淀中特定的DNA序列的浓缩能够通过standard PCR、quantitative real-time PCR或 ChIP on chip、sequencing 和cloning techniques的扩增被分析。SimpleChIP® Kit也提供重要的对照去保证一个成功的ChIP实验。该试剂盒包含一个阳性对照Histone H3 Antibody、一个阴性对照Normal Rabbit IgG Antibody和用于PCR的人源和小鼠ribosomal protein L30 (RPL30)基因引物对。Histone H3是一个染色质中心部分，并且穿过基因组结合到许多DNA序列上，包括RPL30位点。因此，Histone H3 Antibody 提供一个普遍的阳性对照，对于任何位点检测都应该比较丰富。

Gel Staining

FIGURE 1. HeLa cells were formaldehyde-crosslinked and chromatin was prepared and digested as described in Section A of protocol. DNA was purified as described in Section B and 10 μl were separated by electrophoresis on a 1% agarose gel (lane 2) and stained with ethidium bromide. Lane 2 shows that the majority of chromatin was digested to 1 to 5 nucleosomes in length (150 to 900 bp).

Chromatin IP

FIGURE 3. Chromatin immunoprecipitations were performed using digested chromatin from HeLa cells and the indicated ChIP-validated antibodies. Purified DNA was analyzed by quantitative real-time PCR, using SimpleChIP® Human RPL30 Exon 3 Primers #7014 (control primer set), SimpleChIP® Human MyoD1 Exon 1 Primers #4490, and SimpleChIP® Human α Satellite Repeat Primers #4486. The amount of immunoprecipitated DNA in each sample is represented as signal relative to the total amount of input chromatin (equivalent to 1).

图3：使用来自HeLa细胞消化的染色质和指明的ChIP-validated antibodies进行染色质免疫沉淀。使用 SimpleChIP® Human RPL30 Exon 3 Primers #7014 (control primer set)、SimpleChIP® Human MyoD1 Exon 1 Primers #4490和SimpleChIP® Human α Satellite Repeat Primers #4486，通过quantitative real-time PCR分析纯化的DNA。在每个样品中免疫沉淀的DNA数量被看作与input chromatin (相当于1)相关的信号。

 

Chromatin IP

FIGURE 3. Chromatin immunoprecipitations were performed using digested chromatin from HeLa cells and the indicated ChIP-validated antibodies. Purified DNA was analyzed by quantitative real-time PCR, using SimpleChIP® Human RPL30 Exon 3 Primers #7014 (control primer set), SimpleChIP® Human MyoD1 Exon 1 Primers #4490, and SimpleChIP® Human α Satellite Repeat Primers #4486. The amount of immunoprecipitated DNA in each sample is represented as signal relative to the total amount of input chromatin (equivalent to 1).

图3：使用来自HeLa细胞消化的染色质和指明的ChIP-validated antibodies进行染色质免疫沉淀。使用 SimpleChIP® Human RPL30 Exon 3 Primers #7014 (control primer set)、SimpleChIP® Human MyoD1 Exon 1 Primers #4490和SimpleChIP® Human α Satellite Repeat Primers #4486，通过quantitative real-time PCR分析纯化的DNA。在每个样品中免疫沉淀的DNA数量被看作与input chromatin (相当于1)相关的信号。

 

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).

chromatin immunoprecipitation (ChIP)实验室一个强大的和通用的技术，它用于检测在细胞的天然染色质中蛋白质-DNA的相互作用(1,2)。该分析通常被用于鉴定与一个基因组的特异区域或对立面有关的多种蛋白质，或者去鉴定基因组的许多一个特定蛋白结合区域(3-6)。它可以用来确定不同蛋白质的特异性招募到一个基因的启动子或去测量在整个基因上一个特定组蛋白修饰的相对数量(3,4)。除了组蛋白之外，ChIP实验通常用来分析转录因子的结合和辅助因子、DNA复制因子和DNA修复蛋白。当进行ChIP实验时，细胞或组织首先用甲醛固定，甲醛是一个可逆的蛋白质-DNA交联试剂，它可保留细胞中蛋白质-DNA相互作用事件(1,2)。细胞被裂解以及染色质被收获，并且使用声波降解法或酶促消化去分裂染色质。然后，使用特异性的特定蛋白质或组蛋白修饰的抗体进行免疫沉淀染色质。任何DNA序列都与蛋白质相关或者感兴趣的组蛋白修饰将与交联染色质的部分共沉淀，以及通过免疫选择过程DNA序列的相对数量可以被丰富。在免疫沉淀之后，蛋白质-DNA交联是可逆的，以及DNA被纯化。Standard PCR或Quantitative Real-Time PCR通常用于检测通过一个蛋白质特异的免疫沉淀的DNA序列相对数量(1,2)。二者择一地，ChIP分析能够结合 genomic tiling micro-array (ChIP on chip)技术、高通量测序或克隆，所有这些都能进行蛋白质-DNA相互作用的全基因组分析和组蛋白修饰