OneDay ChIP Kit Instruction Manual
Association between proteins and DNA is crucial for many vital cellular functions such as gene transcription and epigenetic silencing. It is important to know the genomic targets of DNA-binding proteins and the mechanisms by which they control and guide gene regulation pathways and cellular proliferation.
Chromatin immunoprecipitation (ChIP) is a technique allowing the analysis of the association of proteins with specific genomic regions in the context of intact cells. ChIP is used to determine changes in epigenetic signatures, chromatin remodeling and transcription regulator recruitments (1). The steps of this technique are cell fixation, chromatin shearing, immunoselection, immunoprecipitation and analysis of the immunoprecipitated DNA. In brief, cells are fixed with a reversible cross-linking agent. Next, the cross-linked chromatin (DNA-Protein) is sheared and the DNA fragments associated with the protein of interest are immunoprecipitated (IP’d) using specific antibodies. Finally, the immunoprecipitated DNA is examined for the presence of particular sequences by quantitative polymerase chain reaction (qPCR). Enrichment of specific sequences in the precipitate indicates that the sequences are associated with the protein of interest in vivo.
The most widely used approach to fix DNA-Protein interactions in the living cell is by formaldehyde fixation (cross-linking) that generates covalent bonds between amino or imino groups of proteins and nucleic acids (2). The formaldehyde cross-links DNA-Protein as well as protein-protein complexes in situ. Following cross-linking, chromatin needs to be sheared very effectively into homogeneous small fragments that can subsequently be used in immunoprecipitation (IP). The Bioruptor TM from Diagenode provides you with high quality sheared chromatin ready-to-ChIP. Moreover, at Diagenode one Shearing module is available and brings an easy and highly reproducible shearing method. Then, antibody binding beads and specific ChIP-grade antibodies are necessary to precipitate the proteins cross-linked to genomic DNA fragments. Finally, the relative amount of a particular DNA fragment specifically IP’d is determined by quantitative PCR as a measure of the occupancy of the protein at that particular position in the genome.
Although ChIP is a very versatile tool, the procedure requires tedious optimization of several reaction conditions. Diagenode provides kits with optimized reagents and simplified protocols for ChIP. Another major drawback to ChIP assays is that the traditional ChIP method is time consuming. It involves two overnight incubations (first for antibody binding to the target and then for the purification of the IP’d DNA). Therefore Diagenode offers kits not only for the traditional ChIP method (such as the Red and Orange ChIP kits) but also for the rapid ChIP method (OneDay ChIP kit).
In the OneDay ChIP method the protocol has been improved to enhance the utility of the ChIP procedure, allowing you to perform many more ChIPs per day and per week. All the procedure can be performed in a day of work as the two overnight incubations have been eliminated. Two major steps have been greatly shortened: 1) the antibody binding is accelerated by incubating antibody with chromatin in an ultrasonic bath (3) and 2) the DNA purification is rapid as it has been simplified by the use of a DNA purifying slurry and does not require multiple steps (see kit overview). There are also two kit formats available: one for 60 IPs and another for 180 IPs.
Get pdf OneDay ChIP Kit Instruction Manual
