Chromatin Regulation / Acetylation
| CSTコード |
包装 |
希望納入価格 (円) |
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| #9002S | 1 Kit | 90,000 | |
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ChIP Kit抗体製品一覧
9002 の推奨プロトコール
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推奨プロトコールはCST社内試験の徹底的なバリデーションに基づいて作成されておりますので、正確かつ再現性の高い結果が得られます。
注：各製品に最適化されたプロトコールをリンクしています。
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9002:
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ChIP (Agarose Beads)
| 特異性・感度 | |
| ChIP 用のすべての抗体にご使用いただけます。 |
SimpleChIP® Enzymatic Chromatin IP Kit (Agarose Beads) (Box 1)
| キット内容 |
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| 製品 | 容量 | 貯法 |
| Glycine Solution (10X) | 100 mL | 4℃ |
| Buffer A (4X) | 25 mL | 4℃ |
| Buffer B (4X) | 25 mL | 4℃ |
| ChIP Buffer (10X) | 20 mL | 4℃ |
| ChIP Elution Buffer (2X) | 7 mL | 4℃ |
| 5 M NaCl | 3 mL | 4℃ |
| 0.5 M EDTA | 1 mL | 4℃ |
| ChIP-Grade Protein G Agarose Beads #9007 | 1 mL | 4℃ |
| DNA Binding Buffer | 30 mL | 室温 |
| DNA Wash Buffer (add 24 mL ethanol before use) | 6 mL | 室温 |
| DNA Elution Buffer | 2 x 1 mL | 室温 |
| DNA Purification Columns | 36 Pack | 室温 |
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SimpleChIP® Enzymatic Chromatin IP Kit (Agarose Beads) (Box 2)
| キット内容 |
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| 製品 | 容量 | 貯法 |
| Protease Inhibitor Cocktail (200X) | 500 μL | -20℃ |
| RNAse A (10 mg/mL) | 50 μL | -20℃ |
| Micrococcal Nuclease (2000 gel units/μL) | 60 μL | -20℃ |
| Proteinase K (20 mg/mL) | 100 μL | -20℃ |
| SimpleChIP™ Human RPL30 Exon 3 Primers #7014 | 150 μL | -20℃ |
| SimpleChIP™ Mouse RPL30 Intron 2 Primers #7015 | 150 μL | -20℃ |
| Histone H3 (D2B12) XP® Rabbit mAb (ChIP Formulated) #4620 | 100 μL | -20℃ |
| Normal Rabbit IgG (1 μg/μL) #2729 | 50 μL | -20℃ |
| 1M DTT | 200 μL | -20℃ |
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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 2. Chromatin immunoprecipitations were performed using digested chromatin from HeLa cells and either Histone H3 (D2B12) XP® Rabbit mAb (ChIP Formulated) #4620 (lane 2), Rpb1 CTD (4H8) Mouse mAb #2629 (lane 3), Di-Methyl Histone H3 (Lys9) Antibody #9753 (lane 4) or Normal Rabbit IgG #2729 (lane 5). Purified DNA was analyzed by standard PCR methods using SimpleChIP® Human RPL30 Exon 3 Primers #7014, SimpleChIP® Human MyoD1 Exon 1 Primers #4490, and SimpleChIP® Human α Satellite Repeat Primers #4486. PCR products were observed for each primer set in the input sample (lane 1) and various protein-specific immunoprecipitations but no PCR products were observed with immunoprecipitation using Normal Rabbit IgG #2729 (lane 5).
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).
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).
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Orlando, V. (2000) Trends Biochem Sci 25, 99-104.
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Kuo, M.H. and Allis, C.D. (1999) Methods 19, 425-33.
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Agalioti, T. et al. (2000) Cell 103, 667-78.
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Soutoglou, E. and Talianidis, I. (2002) Science 295, 1901-4.
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Mikkelsen, T.S. et al. (2007) Nature 448, 553-60.
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Lee, T.I. et al. (2006) Cell 125, 301-13.
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Weinmann, A.S. and Farnham, P.J. (2002) Methods 26, 37-47.
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Wells, J. and Farnham, P.J. (2002) Methods 26, 48-56.
