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 not in the immunoprecipitation using Normal Rabbit IgG #2729 (lane 5).
Chromatin immunoprecipitations were performed using digested chromatin from HeLa cells and the indicated antibodies. Purified DNA was analyzed by quantitative real-time PCR, using SimpleChIP® Human RPL30 Exon 3 Primers #7014, SimpleChIP® Human MyoD1 Exon 1 Primers #4490, and SimpleChIP® Human α Satellite Repeat Primers #4486. The relative abundance of each DNA sequence enriched by protein-specific immunoprecipitations is compared to the amount of the same DNA sequence enriched by the non-specific Normal Rabbit IgG #2729 (background).
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.
Supplied in PBS (pH 7.2), 0.05% Tween® 20, 0.1% BSA, and 0.05% sodium azide. Store at 4°C. This product is stable for 12 months.
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. These beads are compatible with ChIP-seq.
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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