Product Pathways - Chromatin IP
SimpleChIP® Human SUB1 Promoter Primers #5156
|5156S||500 µl ( 250 PCR reactions )||￥2,644.00 现货查询||购买询价|
|5156||carrier free & custom formulation / quantity||email request|
Species cross-reactivity is determined by western blot.
Applications Key: ChIP=Chromatin IP,
SimpleChIP® Human SUB1 Promoter Primers contain a mix of forward and reverse PCR primers that are specific to a region of the human SUB1 promoter. 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 SUB1 gene encodes a transcriptional coactivator and is a direct target of IRF-4.
SimpleChIP™ Human SUB1 Promoter Primers包含正向和反向PCR引物，他们特异性针对人类SUB1启动子的一个区域。该引物可以用来扩增染色质免疫沉淀后的DNA。该引物已经采用SYBR ® Green quantitative real-time PCR优化，并且已经得到联合CST公司的SimpleChIP ® Enzymatic Chromatin IP Kits #9002 and #9003, 和 ChIP-validated antibodies验证。 SUB1基因编码转录辅因子，并且它是IRF4的直接靶基因。
SimpleChIP® Human SUB1 Promoter Primers were tested on DNA isolated from cross-linked cells using the SimpleChIP® Enzymatic Chromatin IP Kit (Magnetic Beads) #9003. Real-time PCR was performed in duplicate on a serial dilution of 2% total input DNA (20 ng, 4 ng, 0.8 ng, and 0.16 ng) using a real-time PCR detection system and SYBR® Green reaction mix. The PCR amplification efficiency (E) and correlation coefficient (R2) were calculated based on the corresponding threshold cycle (CT) of each dilution sample during 40 cycles of real-time PCR (95°C denaturation for 15 sec, 65°C anneal/extension for 60 sec).
PCR product melting curves were obtained for real-time PCR reactions performed using SimpleChIP® Human SUB1 Promoter Primers. Data is shown for both duplicate PCR reactions using 20 ng of total DNA. The melt curve consists of 80 melt cycles, starting at 55°C with increments of 0.5°C per cycle. Each peak is formed from the degradation of a single PCR product.从采用SimpleChIP™ Human SUB1Promoter Primers的实时定量PCR中得到PCR溶解曲线。数据是采用20ng总DNA两次PCR得到的。溶解曲线包括80个循环，起始温度位55度，每个循环增加0.5度。单一PCR产物的降解形成一个峰值。
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.
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)。SimpleChIP® 引物已采用实时定量PCR扩增Chip相关DNA得到优化，并且为确定成功的Chip实验提供了阳性和阴性对照。
- Orlando, V. (2000) Trends Biochem Sci 25, 99-104.
- Kuo, M.H. and Allis, C.D. (1999) Methods 19, 425-33.
- Agalioti, T. et al. (2000) Cell 103, 667-78.
- Soutoglou, E. and Talianidis, I. (2002) Science 295, 1901-4.
- Mikkelsen, T.S. et al. (2007) Nature 448, 553-60.
- Lee, T.I. et al. (2006) Cell 125, 301-13.
- Weinmann, A.S. and Farnham, P.J. (2002) Methods 26, 37-47.
- Wells, J. and Farnham, P.J. (2002) Methods 26, 48-56.
Have you published research involving the use of our products? If so we'd love to hear about it. Please let us know!
- 4299 IRF-4 (D43H10) Rabbit mAb
- 4964 IRF-4 Antibody
- 9002 SimpleChIP® Enzymatic Chromatin IP Kit (Agarose Beads)
- 9003 SimpleChIP® Enzymatic Chromatin IP Kit (Magnetic Beads)
- 9006 ChIP-Grade Protein G Magnetic Beads
- 9007 ChIP-Grade Protein G Agarose Beads
For Research Use Only. Not For Use In Diagnostic Procedures.
SYBR is a registered trademark of Life Technologies Corporation.
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.
用户评论 --- 共 0 条