Cell Signaling Technology

Product Pathways - Chromatin Regulation

SimpleChIP™ Enzymatic Chromatin IP Kit (Magnetic Beads) #9003

Kit Includes Volume Storage Temp
Glycine Solution (10X) 100 milliliters 4°C
Buffer A (4X) 25 milliliters 4°C
Buffer B (4X) 25 milliliters 4°C
ChIP Buffer (10X) 20 milliliters 4°C
ChIP Elution Buffer (2X) 7 milliliters 4°C
5 M NaCl 3 milliliters 4°C
0.5 M EDTA 1 milliliters 4°C
ChIP-Grade Protein G Magnetic Beads # 9006 1 milliliters 4°C
DNA Binding Buffer 30 milliliters RT
DNA Wash Buffer(add 24 ml ethanol before use) 6 milliliters RT
DNA Elution Buffer 1 milliliters RT
DNA Purification Columns 36 Pack RT
Protease Inhibitor Cocktail (200X) 500 microliters -20°C
RNAse A (10 mg/ml) 20 microliters -20°C
Micrococcal Nuclease 60 microliters -20°C
Proteinase K 100 microliters -20°C
Human RPL30 Control Primers (5 μM) 150 microliters -20°C
Mouse RPL30 Control Primers (5 μM) 150 microliters -20°C
Histone H3 Antibody (ChIP-Formulated) 100 microliters -20°C
Normal Rabbit IgG # 2729 50 microliters -20°C
1M DTT 200 microliters -20°C

Note: The 6-Tube Magnetic Separation Rack (Product #7017) or a similar magnetic rack is required, but not included in the kit. When using the Magnetic Separation Rack, ChiP-Grade Protein G Magnetic Beads separate to the side of the tube instead of the bottom, eliminating centrifugation steps, minimizing sample loss, and increasing washing efficiency of the immunocomplexes.

Species Cross-Reactivity

H M R Mk

Reactivity Key:  H=Human  M=Mouse  R=Rat  Mk=Monkey

Description

The SimpleChIP™ Enzymatic Chromatin IP Kit (Magnetic Beads) contains the buffers and reagents necessary to perform up to 6 chromatin preparations and 30 chromatin immunoprecipitations and is optimized for 4 X 107 cells per experiment. A complete assay can be performed in as little as two days and can easily be scaled up or down for use with more or fewer cells.Cells are fixed with formaldehyde and lysed, and chromatin is fragmented by partial digestion with Micrococcal Nuclease to obtain chromatin fragments of 1 to 5 nucleosomes. Enzymatic fragmentation of chromatin is much milder than sonication and eliminates problems resulting from variability in sonication power and emulsification of chromatin during sonication, which can result in incomplete fragmentation of chromatin or loss of antibody epitopes due to protein denaturation and degradation. Chromatin immunoprecipitations are performed using ChIP-validated antibodies and ChIP-Grade Protein G Magnetic Beads. After reversal of protein-DNA cross-links, the DNA is purified using DNA purification spin columns, allowing for easy and efficient recovery of DNA and removal of protein contaminants without the need for phenol/chloroform extractions and ethanol precipitations. The enrichment of particular DNA sequences during immunoprecipitation can be analyzed by standard PCR, quantitative real-time PCR, or amplification for ChIP on chip, sequencing or cloning techniques.The SimpleChIP™ Kit also provides important controls to ensure a successful ChIP experiment. The kit contains a positive control Histone H3 Antibody, a negative control Normal Rabbit IgG Antibody and primer sets for PCR detection of the human and mouse ribosomal protein L30 (RPL30) genes. Histone H3 is a core component of chromatin and is bound to most DNA sequences throughout the genome, including the RPL30 locus. Thus, the Histone H3 Antibody provides a universal positive control that should enrich for almost any locus examined.

Specificity / Sensitivity

The SimpleChIP™ Enzymatic Chromatin IP Kit can be utilized with any ChIP-validated antibody to detect endogenous levels of protein-DNA interactions and histone modifications in mammalian cells (see Figures 2 and 3). The positive control Histone H3 Antibody recognizes many different species of the highly conserved Histone H3 protein, including human, mouse, rat and monkey. Primer sets are included for the human and mouse positive control RPL30 gene loci; however, the use of other species with the kit requires the design of additional control primer sets.

Results

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).

FIGURE 2. Chromatin immunoprecipitations were performed using digested chromatin from HeLa cells and either Histone H3 Antibody #2650 (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 primers specific for the transcriptionally active RPL30 gene (control primer set), the inactive MYOD gene, or the heterochromatic alpha satellite repeat element. PCR products were observed for each primer set in the input sample (lane 1) and various ChIP samples, but not in the Normal Rabbit IgG ChIP sample (lane 5).

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 primers specific for the transcriptionally active RPL30 gene (control primer set), the inactive MYOD gene, or the heterochromatic alpha satellite repeat element. The amount of immunoprecipitated DNA in each sample is represented as signal relative to the total amount of input chromatin (equivalent to 1).


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 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.

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