The ChIP (chromatin immunoprecipitation) method has emerged as a very powerful tool to study in vivo protein-DNA interactions. It has been applied to the dynamics of chromatin structure, regulation by transcription factors and co-regulators and other epigenetic changes. The ChIP procedure can be divided into three major steps: 1) isolation and fragmentation of the chromatin after formaldehyde fixation, 2) immunoprecipitation of specific chromatin fragments using antibody against the protein of interest and 3) analysis of immunoprecipitated fractions to determine if target DNA is bound by the protein of interest (evidenced by enrichment of DNA relative to chromatin).
The ChIP method is a complicated and technically demanding procedure, especially when performed with antibodies against transcription factors rather than abundant histone proteins. There are multiple steps that need to be optimized, including the choice of antibody, the conditions for shearing chromatin using sonication and the analysis of
immunoprecipitated DNA. The ChIP-IT Kit from Active Motif provides a complete solution to all potential problems associated with the ChIP method.
The Active Motif ChIP-IT Kit comes with all the reagents needed for 25 reactions of the ChIP assay. In addition, it provides a detailed manual which saves time for protocol optimization. The protocol in the manual is easy to follow, even for a novice. The biggest advantage of the ChIP-IT kit is that it contains both positive (anti-TFIIB) and negative (negative IgG) control antibodies for the ChIP. This feature enables the user to determine if results are due to ChIP or the antibody, as well as determining if the ChIP assay has errors. The kit also supplies PCR primers for both the positive and negative controls, facilitating the analysis of ChIP DNA by PCR. The column purified ChIP DNA can be directly used for downstream applications, either genome-wide ChIP-chip analysis or selective quantitative real-time PCR assay.
The Active Motif ChIP-IT Kit is a nice kit for the ChIP assay. Though the price of the kit is a little more expensive than other commercially available ChIP kits, it is well worth the money. The newer version of the kit gives the user more choices with regard to assay control and method of shearing the chromatin. For first time users, I strongly recommend starting with the full kit using the provided controls and sonication directions as suggested, initially.
Fan Yang, Ph.D.
Staff Scientist
Atlantic Biolabs