Chromatin immunoprecipitation 1188840 215112068 2008-05-26T19:31:25Z Modify 380367 rm comma '''Chromatin immunoprecipitation''' ('''ChIP''') [[assay]] is a method used for experiments in [[molecular biology]]. The purpose of this assay is to determine whether [[protein]]s including (but not limited to) [[transcription factors]] bind to a particular region on the [[endogenous]] [[chromatin]] of living [[cell (biology)|cell]]s or tissues. The ''[[in vivo]]'' nature of this method is in contrast to other approaches traditionally employed to answer the same questions. The principle underpinning this assay is that [[DNA]]-bound proteins (including transcription factors) in living cells can be [[cross-link]]ed to the [[chromatin]] where they are situated. This is usually accomplished by a gentle [[formaldehyde]] fixation, although it is sometimes advantageous to use the reversible crosslinker DTBP instead. Following fixation, the cells are [[Lysis|lysed]] and the DNA is broken into pieces 0.2-1 kb in length by [[sonication]]. Once the proteins are immobilized on the chromatin and the chromatin is fragmented, whole protein-DNA complexes can be [[immunoprecipitated]] using an [[antibody]] specific for the protein in question. The DNA from the isolated protein/DNA fraction can then be purified. The identity of the DNA fragments isolated in complex with the protein of interest can then be determined by [[PCR]] using primers specific for the DNA regions that the protein in question is hypothesized to bind. Alternatively, when one wants to find where the protein binds across the whole [[genome]], a [[DNA microarray]] can be used ([[ChIP-on-chip|ChIP-on-chip or ChIP-chip]]) allowing for the characterization of the [[cistrome]]. As well, [[Chip-Sequencing|ChIP-Sequencing]] has recently emerged as a new technology that can localize protein binding sites in a high-throughput, cost-effective fashion. ==Major disadvantages and solutions== The major disadvantage is the requirement for highly specific antibodies for each protein to be tested. This can be overcome by the construction of proteins fused to either [[epitopes]] (like HA or c-myc) recognized by antibodies widely available, or [[amino acid]] sequences recognized by [[enzymes]] that add [[biotin]] to some residues. Biotin has the great advantage of binding with extremely high [[chemical affinity|affinity]] and specificity to the proteins [[avidin]], [[streptavidin]] and [[NeutrAvidin]]. ==External links== * {{MeshName|Chromatin+immunoprecipitation}} *[http://www.cshprotocols.org/cgi/content/full/2007/12/pdb.prot4767 Chromatin Immunopreciptation (ChIP) on Unfixed Chromatin from Cells and Tissues to Analyze Histone Modifications] *[http://www.cshprotocols.org/cgi/content/full/2006/23/pdb.prot4560 Chromatin Immunoprecipitation (ChIP) of Protein Complexes: Mapping of Genomic Targets of Nuclear Proteins in Cultured Cells] {{Immunologic techniques and tests}} [[Category:Protein methods]] [[de:Chromatin-Immunopräzipitation]] [[fr:Chip on chip]] [[nl:Chromatine-immunoprecipitatie]] [[ja:クロマチン免疫沈降]]