Product Pathways - Chromatin Regulation / Epigenetics
SimpleChIP® Plus Enzymatic Chromatin IP Kit (Agarose Beads) #9004
|Kit Includes||Quantity||Applications||Reactivity||Homology†||MW (kDa)||Isotype|
|DNA Binding Buffer||30 ml|
|DNA Wash Buffer (add 4x volume ethanol before use)||6 ml|
|DNA Elution Buffer||1 ml|
|DNA Purification Columns and Collection Tubes||36 Pack|
|Micrococcal Nuclease #10011||60 µl|
|Proteinase K||100 µl|
|Histone H3 (D2B12) XP® Rabbit mAb (ChIP Formulated) #4620||100 µl||R,Hm,Mk,C,Dm,X,Z,B,||17||Rabbit IgG|
|Glycine Solution (10X)||100 ml|
|Buffer A (4X)||25 ml|
|Buffer B (4X)||25 ml|
|ChIP Buffer (10X)||20 ml|
|ChIP Elution Buffer (2X)||7 ml|
|5 M NaCl||3 ml|
|0.5 M EDTA, pH 8.0 #7011||1 ml|
|Protease Inhibitor Cocktail (200X)||750 µl|
|RNAse A (10 mg/ml)||50 µl|
|SimpleChIP® Human RPL30 Exon 3 Primers #7014||150 µl|
|SimpleChIP® Mouse RPL30 Intron 2 Primers #7015||150 µl|
|DTT (Dithiothreitol) #7016||192.8 mg||154.25 g/mol|
|ChIP-Grade Protein G Agarose Beads #9007||1 ml|
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.
SimpleChIP® Plus Enzymatic Chromatin IP Kit与任何ChIP-validated antibody一起被利用去检测内源性的蛋白质-DNA相互作用和哺乳动物细胞和组织中组蛋白修饰(见图1和2)。阳性对照Histone H3 Antibody可检测高度保守的Histone H3蛋白的许多不同物种，包括人、小鼠、大鼠和猴。引物对包含人源和小鼠阳性对照RPL30基因位点；然而，该试剂盒中其它物种的使用需要额外设计对照引物。
The SimpleChIP® Plus Enzymatic Chromatin IP Kit (Agarose Beads) 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 standard PCR, quantitative real-time PCR, or amplification for ChIP on chip, sequencing or cloning techniques. 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.
SimpleChIP® Plus Enzymatic Chromatin IP Kit (Agarose Beads)包含缓冲液和试剂，细胞和组织样品必须能进行30次染色质免疫沉淀，并且每次免疫沉淀实验最好需要4 X 106细胞数或25 mg组织。一个完整的实验至少两天时间被完成，并且对于细胞数或组织样品使用多或少也很容易扩大或缩小试剂量。细胞和组织使用甲醛固定并裂解，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非常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DNA，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® Plus 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，该对照应该是对于任何位点检测都要丰富。
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 cross-linked and disaggregated into a single-cell suspension using a Dounce homogenizer. The chromatin was prepared and digested, and 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).
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。
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(5-8)。
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- Wang, J.D. et al. (2015) Autophagy 11, 2057-2073.Applications:
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- 12931 Nuclease-free Water
- 14231 SimpleChIP® Chromatin IP Buffers
- 14282 SimpleChIP® Cell Lysis Buffers A & B
- 2729 Normal Rabbit IgG
- 4620 Histone H3 (D2B12) XP® Rabbit mAb (ChIP Formulated)
- 7014 SimpleChIP® Human RPL30 Exon 3 Primers
- 7015 SimpleChIP® Mouse RPL30 Intron 2 Primers
- 9007 ChIP-Grade Protein G Agarose Beads
For Research Use Only. Not For Use In Diagnostic Procedures.
Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.
SimpleChIP is a registered trademark of Cell Signaling Technology, Inc.
Cell Signaling Technology® is a trademark of Cell Signaling Technology, Inc.
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