ChIP-Grade Protein G Magnetic Beads

• 产品编号：CST-9006S      品牌：CST       原厂货号：9006S
• 产品分类：生化和细胞分析 > 微珠和磁珠 > 配体耦联磁珠 > Protein A&G 磁珠
• 应用分类：

描述：

ChIP级别的蛋白G磁珠是在染色质共沉淀（Chip）中用于小规模分离免疫复合物。重组蛋白G以其截断形式与无孔磁性颗粒相偶联，表现出与很多物种（包括人类、兔子、小鼠、大鼠和养）的IgG亚型的高亲和力，并且可以与这些抗体共同应用于免疫沉淀实验。磁珠保存在包含BSA（1mg/ml)的缓冲液中，可以在分离免疫复合物过程中减少蛋白和DNA的非特异性结合。用我们的6管磁性分离架（产品信息为#7017）可以将磁珠从溶液中分离出来。后者可以将磁珠聚集在管底的一侧，使得离心的步骤减少，减少了样本损失，液提高了清洗效率。

短暂涡旋管子以重悬微珠，每一个染色质免疫沉淀反应加入30ul微球。对于清洗微球和随后的洗脱免疫复合物，微球可以用我们的6管磁性分离架（产品信息为#7017）从溶液中分离。将很有微珠的管子置于磁性分离架上#7017，在移除上清前等待1-2分钟。将管子从磁性分离架移开，采用轻轻涡旋或者摇摆管子的方法用新的溶液重悬微珠。

染色质免疫共沉淀(Chip)分析用来探测天然染色质上的细胞中蛋白和DNA相互作用的强大和灵活的技术(1、2)。这种分析方法可以用来确定多个与特定的区域的基因组,或相反的相关的蛋白质，识别特定蛋白结合的基因组的许多区域 (3 - 6)。它可以用于确定招募到基因启动子的各种蛋白质的特定顺序或“测量”整个基因位点发生特定的组蛋白修饰的相对量 (3、4)。除了组蛋白，Chip可用于分析结合的的转录因子和共因子、DNA复制因子和DNA修复蛋白。 　　当进行Chip时，首先用甲醛固定细胞或组织、一种蛋白和DNA可逆交联剂，可以保护”发生在细胞中的蛋白和DNA的相互作用 (1、2)。裂解细胞获得染色质，用超声破碎或酶消化。然后染色质与针对于特定的蛋白质或组蛋白修饰的抗体发生免疫沉淀。任何与目的蛋白或组蛋白修饰相关的DNA序将作为交联的染色质复合体的一部分发生共沉淀，DNA序列的相对量将经过免疫选择过程富集。免疫沉淀后，蛋白和DNA交联发生解交联，然后纯化DNA。标准PCR或实时定量PCR可以用来测量经蛋白特一的免疫沉淀浓缩DNA序列的进行定量 (1、2)。另外,该Chip可以与基因组芯片（Chip on Chip)技术、高通量测序、或者克隆技术相结合，它们可以对蛋白和DNA的相互作用及组蛋白修饰做全基因组分析 (5 - 8)。

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

ChIP-Grade Protein G Magnetic Beads are an affinity matrix for the small-scale isolation of immunocomplexes from chromatin immunoprecipitation (ChIP) assays. A truncated form of recombinant protein G is covalently coupled to a nonporous paramagnetic particle. Protein G exhibits high affinity for subclasses of IgG from many species (including human, rabbit, mouse, rat and sheep) and can be used for immunoprecipitation assays with these antibodies. The beads are stored in buffer containing BSA (1 mg/ml) to block non-specific binding of proteins and DNA during isolation of immunocomplexes. Beads can be separated from solution using our 6-Tube Magnetic Separation Rack #7017, which concentrates the beads to the side of the tube instead of the bottom. This eliminates centrifugation steps, minimizes sample loss and increases washing efficiency.

Directions for Use

Vortex tube briefly to resuspend the beads. Add 30 μl of bead slurry to each chromatin immunoprecipitation (ChIP) reaction. For bead washing and subsequent elution of immunocomplexes, the beads can be separated from solution using our 6-Tube Magnetic Separation Rack #7017. Place the tubes containing the beads in the Magnetic Separation Rack and wait 1 to 2 minutes for the solution to clear before carefully removing the supernatant. Remove the tubes from the Magnetic Separation Rack, add new solution and resuspend the beads by gently vortexing or rocking the tube.

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).

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 PBS Buffer (pH 7.2), 0.05% Tween®20,
1mg/ml BSA, and 0.02% sodium azide. Store at 4ºC.