SimpleChIP? Human γ-Actin Intron 3 Primers

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• 产品分类：PCR/RT-PCR/qRT-PCR > 其它 > 引物 > 其他引物与寡核苷酸
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描述：

SimpleChIP^® Human γ-Actin Intron 3 Primers包含一个正向和反向的PCR混合引物，该对引物是特异性人源γ-actin基因第3内含子的引物。这些引物对通常扩增C和IP实验中分离出的DNA 。该引物对在SYBR^® Green quantitative real-time PCR使用中已经是最佳引物，并且在SimpleChIP^® Enzymatic Chromatin IP Kits #9002 and #9003 和ChIP-validated antibodies from Cell Signaling Technology^® 的结合中被检测。 γ-actin基因在所有的细胞中广泛的转录，同时发现在激活的基因第3内含子是富含有组蛋白修饰，例如组蛋白 H3 Lys36 三甲基化和组蛋白 H2B Lys120 泛素化。

在细胞内天然染色质的环境下对于探索蛋白质-DNA相互作用，染色质免疫沉淀（chromatin immunoprecipitation ，ChIP）分析是一个强大的和通用的技术(1,2)。该实验通常用于鉴定多种蛋白与基因组的一个特定区域结合或者鉴定基因组的多个区域被一个特定的蛋白结合(3-6)。ChIP通常确定不同蛋白质招募到一个基因的启动子的特异的序列或者测量在整个基因位点上的特异组蛋白修饰的相对数量(3,4)。除了组蛋白，ChIP实验通常用于分析转录因子与辅助因子、DNA复制因子和DNA修复因子的结合能力。当进行ChIP 实验，细胞首先用甲醛固定，甲醛是一个可逆的蛋白质-DNA交联剂，它可保存蛋白质-DNA在细胞内相互作用 (1,2)。细胞被裂解，并且染色质提取并使用超声或酶促消化成片段。然后片段染色质使用特定蛋白质或组蛋白修饰的特异的抗体进行免疫沉淀反应。与蛋白质或感兴趣组蛋白修饰的任何DNA序列将作为交联染色质复合物的一部分共沉淀，并且通过免疫选择过程相对数量的DNA序列将增加。在免疫沉淀之后，蛋白质-DNA交联保留下来且DNA被纯化。标准PCR或定量real-time PCR经常被用于测量通过一个蛋白质特定免疫沉淀的DNA序列的富集量(1,2)。另外，ChIP实验能与基因组微阵列技术 (ChIP on chip)、高通量测序技术(ChIP-Seq)或克隆策略结合，它们每一个都允许蛋白质-DNA相互作用和组蛋白修饰的全基因组分析(5-8)。使用real-time quantitative PCR，SimpleChIP^® primers已经是ChIP分离的DNA的扩增最好的引物，并且能够提供重要的阳性对照和阴性对照，这些对照通常用于确认一个成功的ChIP实验。

1. Orlando, V. (2000) Trends Biochem Sci 25, 99-104.
2. Kuo, M.H. and Allis, C.D. (1999) Methods 19, 425-33.
3. Agalioti, T. et al. (2000) Cell 103, 667-78.
4. Soutoglou, E. and Talianidis, I. (2002) Science 295, 1901-4.
5. Mikkelsen, T.S. et al. (2007) Nature 448, 553-60.
6. Lee, T.I. et al. (2006) Cell 125, 301-13.
7. Weinmann, A.S. and Farnham, P.J. (2002) Methods 26, 37-47.
8. Wells, J. and Farnham, P.J. (2002) Methods 26, 48-56.

原厂资料：

Description

SimpleChIP^® Human γ-Actin Intron 3 Primers contain a mix of forward and reverse PCR primers that are specific to intron 3 of the human γ-actin gene. These primers can be used to amplify DNA that has been isolated using chromatin immunoprecipitation (ChIP). Primers have been optimized for use in SYBR® Green quantitative real-time PCR and have been tested in conjunction with SimpleChIP® Enzymatic Chromatin IP Kits #9002 and #9003 and ChIP-validated antibodies from Cell Signaling Technology®. The γ-actin gene is actively transcribed in all cell types and the intron 3 region is highly enriched for histone modifications found in the body of active genes, such as histone H3 Lys36 tri-methylation and histone H2B Lys120 ubiquitination.

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 either identify multiple proteins associated with a specific region of the genome or to identify the many regions of the genome bound by a particular protein (3-6). ChIP 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 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. Fragmented 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 are often 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 (ChIP-Seq), or cloning strategies, all of which allow for genome-wide analysis of protein-DNA interactions and histone modifications (5-8). SimpleChIP® primers have been optimized for amplification of ChIP-isolated DNA using real-time quantitative PCR and provide important positive and negative controls that can be used to confirm a successful ChIP experiment.

1. Orlando, V. (2000) Trends Biochem Sci 25, 99-104.
2. Kuo, M.H. and Allis, C.D. (1999) Methods 19, 425-33.
3. Agalioti, T. et al. (2000) Cell 103, 667-78.
4. Soutoglou, E. and Talianidis, I. (2002) Science 295, 1901-4.
5. Mikkelsen, T.S. et al. (2007) Nature 448, 553-60.
6. Lee, T.I. et al. (2006) Cell 125, 301-13.
7. Weinmann, A.S. and Farnham, P.J. (2002) Methods 26, 37-47.
8. Wells, J. and Farnham, P.J. (2002) Methods 26, 48-56.

注意事项：

Storage: Supplied in nuclease-free water at a concentration of
5 μM (each primer is at a final concentration of 5 μM). Store at
-20°C.