The SimpleChIP® Plus Sonication Chromatin IP Kit contains the buffers and reagents necessary to perform up to 24 chromatin immunoprecipitations from cells or tissue samples, and is optimized for 4 X 106 cells or 25 mg of tissue per immunoprecipitation. 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 less cells or tissue sample. This kit is compatible with both ChIP-qPCR and ChIP-seq.
Cells or tissue are fixed with formaldehyde and lysed, and chromatin is fragmented by sonication to obtain chromatin fragments ranging from 200 to 1000 bp. Sonication-based fragmentation is the more traditional method for fragmenting chromatin; however, sonication should be optimized such that the desired fragment size is achieved with the minimal amount of sonication required, as over-sonication can result in a decrease in immunoprecipitation, specifically for transcription factors and cofactors. The cell and nuclear lysis buffers for this kit have been optimized to maximize enrichment of histones, transcription factors and cofactors. 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 a variety of methods, including standard PCR, quantitative real-time PCR, or amplification for ChIP on chip, sequencing or cloning techniques.
The SimpleChIP® Plus 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.
® Plus Sonication Chromatin IP Kit can be utilized with ChIP-validated antibodies to detect endogenous levels of protein-DNA interactions and histone modifications in mammalian cells and tissue samples (see Figures 1 to 4). The cell and nuclear lysis buffers for this kit have been optimized to maximize enrichment of chromatin containing histones, transcription factors and cofactors. 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.