Chromatin is comprised of nucleosome subunits, each of which consists of DNA wound around several histone proteins. Recent findings have revealed that large multiprotein complexes are often involved in regulatory processes taking place in the nucleus. The regulation of transcription in eukaryotic cells is critically dependent on the dynamic state of chromatin. Transcriptional activation and inactivation is intimately associated with the either the relaxed or taut conformation of chromatin structure. To unravel the structure and dynamics of these nuclear protein complexes and determine their interactions with the DNA template, many scientists use an assay known as the chromatin immunoprecipitation (ChIP) assay. The ChIP assay combines two straightforward steps — first, in vivo formaldehyde cross-linking of whole cells that freezes protein-protein and protein-DNA interactions, followed by immunoprecipitation of protein-DNA complexes with specific antibodies from sonicated extracts. Scientists interested in performing this assay should consider using the Chromatin Immunoprecipitation (ChIP) Assay Kit from Upstate Biotechnology. The kit is a collection of several key reagents (ten in total) that assist in various points during the ChIP protocol.
The ChIP Assay Kit provides the user with a number of the important solutions for this assay but users still need to optimize a number of key steps on their own, and there are other required reagents that are not provided. For instance, users will need to optimize the length of time for the formaldehyde treatment and the sonication step that shears the DNA. Also, they will obviously need to use their antibody of choice for precipitating the protein/DNA complexes. Once the assay has been performed, users must also design their own methods to analyze what has been immunoprecipitated (i.e., either by quantitative PCR or Southern slot-blot analysis). Nevertheless, prospective buyers who have never performed the ChIP assay before will be pleased to know that the kit comes with a supplied lysis buffer to lyse formaldehyde-treated cells prior to sonication, a protein A agarose slurry that precipitates antibody-protein-DNA complexes, several wash buffers that are necessary for reducing non-specific background interactions, and a 5M NaCl solution that reverses the formaldehyde cross-links. A complete ChIP protocol is provided, and is also available on the Upstate Biotechnology website, but many scientists have designed their own ChIP protocols to suit their needs.
I found the ChIP kit to be useful for getting a quick start on the ChIP assay. There are many steps in the protocol, and having some of the often-used solutions already in hand makes the cumbersome assay more user-friendly.
Joe Lau
Graduate student
Immunobiology Center
Mount Sinai School of Medicine