SimpleChIP^® Enzymatic Chromatin IP Kit (Agarose Beads)

Please store components at the temperatures indicated on the individual tube labels.

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.

The SimpleChIP^® Enzymatic Chromatin IP Kit (Agarose Beads) #9002 contains the buffers and reagents necessary to perform up to 6 chromatin preparations and 30 chromatin immunoprecipitations and is optimized for 4 X 10⁶ 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 Agarose 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 a variety of methods, including standard PCR and quantitative real-time PCR. Please note that this kit is not compatible with ChIP-seq because the ChIP-Grade Protein G Agarose Beads are blocked with sonicated salmon sperm DNA, which interferes with downstream sequencing.
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.