CST的SimpleChIP? 酶解染色質免疫沉淀試劑盒是由CST與New England Biolabs的科學家聯合開發的，因此包含了最高質量的研究試劑。該試劑盒有兩種：Protein G 瓊脂糖微球和Protein G 磁珠，所包含的緩沖液和試劑足夠做30次ChIP實驗。Protein G 瓊脂微球和Protein G 磁珠也有單獨的產品。

酶消化染色質比超聲破碎更加溫和。溫和的處理方式更好保留了染色質完整性和蛋白的抗原表位， 增加了免疫沉淀的效率。

詳細的操作步驟專為細胞和組織樣品優化，節約您的時間和試劑。

試劑盒可與任何ChIP級抗體一起檢測哺乳動物細胞內源水平的蛋白質-DNA相互作用和組蛋白修飾。

包含所有試劑和對照，節約時間，提供合適的實驗對照。

試劑盒內的試劑在公司內部用于驗證我們的ChIP 抗體，將方便您的優化。

試劑盒內包含的ChIP 級磁珠非常適于下游的ChIP-on-chip 或 ChIP 測序，因為其更高的靈敏度和低背景。

9002?????? SimpleChIP? Enzymatic Chromatin IP Kit (Agarose Beads)????

?

9003?????? SimpleChIP? Enzymatic Chromatin IP Kit (Magnetic Beads)

SimpleChIP? Enzymatic Chromatin IP Kit (Agarose Beads)包含緩沖液和試劑，必須進行6次染色質準備和30次染色質免疫沉淀，并且每次實驗最佳的細胞數為4 X 107 。一個完整的實驗至少兩天時間被完成，并且對于使用多的或少的細胞數試劑盒很容易擴大或縮小。細胞使用甲醛固定，并且通過微球菌核酸酶部分程度上酶切染色質成片段，從而獲得1-5個核小體的染色質片段。染色質的酶切片段是比超聲更加溫和以及消除了由超聲功率的可比性以及在超聲講解中染色質的乳化作用帶來的問題，這能夠導致染色質的不完全片段化或由于蛋白質變性和降解造成的抗體表位的損失。使用ChIP-validated antibodies和ChIP-Grade Protein G Agarose Beads進行染色質免疫沉淀。在蛋白質-DNA交聯的逆轉之后，使用DNA純化旋轉柱被純化，這允許DNA更容易和高效的恢復，并且蛋白質的移除不需要苯酚/氯仿的提取和乙醇的沉淀。在免疫沉淀中特定的DNA序列的濃縮能夠通過standard PCR、quantitative real-time PCR或 ChIP on chip、sequencing 和cloning techniques的擴增被分析。SimpleChIP? Kit也提供重要的對照去保證一個成功的ChIP實驗。該試劑盒包含一個陽性對照Histone H3 Antibody、一個陰性對照Normal Rabbit IgG Antibody和用于PCR的人源和小鼠ribosomal protein L30 (RPL30)基因引物對。Histone H3是一個染色質中心部分，并且穿過基因組結合到許多DNA序列上，包括RPL30位點。因此，Histone H3 Antibody 提供一個普遍的陽性對照，對于任何位點檢測都應該比較豐富。

?

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).

圖3：使用來自HeLa細胞消化的染色質和指明的ChIP-validated antibodies進行染色質免疫沉淀。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，通過quantitative real-time PCR分析純化的DNA。在每個樣品中免疫沉淀的DNA數量被看作與input chromatin (相當于1)相關的信號。

?

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).

圖3：使用來自HeLa細胞消化的染色質和指明的ChIP-validated antibodies進行染色質免疫沉淀。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，通過quantitative real-time PCR分析純化的DNA。在每個樣品中免疫沉淀的DNA數量被看作與input chromatin (相當于1)相關的信號。

?

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)實驗室一個強大的和通用的技術，它用于檢測在細胞的天然染色質中蛋白質-DNA的相互作用(1,2)。該分析通常被用于鑒定與一個基因組的特異區域或對立面有關的多種蛋白質，或者去鑒定基因組的許多一個特定蛋白結合區域(3-6)。它可以用來確定不同蛋白質的特異性招募到一個基因的啟動子或去測量在整個基因上一個特定組蛋白修飾的相對數量(3,4)。除了組蛋白之外，ChIP實驗通常用來分析轉錄因子的結合和輔助因子、DNA復制因子和DNA修復蛋白。當進行ChIP實驗時，細胞或組織首先用甲醛固定，甲醛是一個可逆的蛋白質-DNA交聯試劑，它可保留細胞中蛋白質-DNA相互作用事件(1,2)。細胞被裂解以及染色質被收獲，并且使用聲波降解法或酶促消化去分裂染色質。然后，使用特異性的特定蛋白質或組蛋白修飾的抗體進行免疫沉淀染色質。任何DNA序列都與蛋白質相關或者感興趣的組蛋白修飾將與交聯染色質的部分共沉淀，以及通過免疫選擇過程DNA序列的相對數量可以被豐富。在免疫沉淀之后，蛋白質-DNA交聯是可逆的，以及DNA被純化。Standard PCR或Quantitative Real-Time PCR通常用于檢測通過一個蛋白質特異的免疫沉淀的DNA序列相對數量(1,2)。二者擇一地，ChIP分析能夠結合 genomic tiling micro-array (ChIP on chip)技術、高通量測序或克隆，所有這些都能進行蛋白質-DNA相互作用的全基因組分析和組蛋白修飾