| 货号 | 13653S |
| 反应种属 | Human |
| 应用 | ChIP |
| 使用方法 | 1. Label the appropriate number of PCR tubes or PCR plates compatible with the model of real-time PCR machine to be used. PCR reactions should be performed in duplicate and should include a tube with no DNA to control for contamination, and a serial dilution of a 2% total input chromatin DNA (undiluted, 1:5, 1:25, 1:125), which is used to create a standard curve and determine amplification efficiency. |
| 供应商 | CST |
| 背景 | 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). |
| 存放说明 | -20C |
| 参考文献 | 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. |
SimpleChIP® Human β-Actin 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 β-Actin 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. |
