Chromatin immunoprecipitation
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Chromatin immunoprecipitation (ChIP) assay is a method used for experiments in molecular biology. The purpose of this assay is to determine whether proteins including (but not limited to) transcription factors bind to a particular region on the endogenous chromatin of living cells 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-linked 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 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 or ChIP-chip) allowing for the characterization of the cistrome. As well, ChIP-Sequencing has recently emerged as a new technology that can localize protein binding sites in a high-throughput, cost-effective fashion.
[edit] 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 affinity and specificity to the proteins avidin, streptavidin and NeutrAvidin.
[edit] External links
- MeSH Chromatin+immunoprecipitation
- Chromatin Immunopreciptation (ChIP) on Unfixed Chromatin from Cells and Tissues to Analyze Histone Modifications
- Chromatin Immunoprecipitation (ChIP) of Protein Complexes: Mapping of Genomic Targets of Nuclear Proteins in Cultured Cells
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