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Chromatin affinity purification.
Methods Mol Biol. 2012;809:237-53
Authors: Harada R, Nepveu A
Abstract
Chromatin immunoprecipitation (ChIP) has become an essential assay in the field of transcriptional regulation. It is currently the most popular method to monitor the in vivo interaction between a protein and specific genomic sites. The method can also serve to identify novel transcriptional targets when the immunoprecipitated chromatin, sometimes called chipped DNA, is used either as a probe in hybridization experiments with microarrays of genomic DNA (ChIP-chip) or as template in DNA sequencing (ChIP-Seq). ChIP assays rely on the availability of good antibodies that can specifically and efficiently immunoprecipitate the protein under study even after cross-linking. However, good antibodies are not always available. To circumvent this problem, we have developed and validated the method of chromatin affinity purification (ChAP). The subsequent microarray analysis is then referred to as ChAP-chip. In brief, the protein under study is expressed together with two tags in order to allow the purification of chromatin by tandem affinity purification. To ensure that only true targets are identified, it is important to express the recombinant tagged-protein at physiological level. This requirement is not trivial as most expression vectors are designed to express proteins at high levels. We found most convenient to use an inducible retroviral vector in the absence of inducer and transactivator protein. We describe the procedure to generate cells stably expressing recombinant tagged-proteins at physiological level and then to purify the associated chromatin by affinity purification. Targets identified in this manner were validated in independent ChAP assays as well as in ChIP assays using antibodies against the endogenous protein.
PMID: 22113280 [PubMed - in process]
More...
Chromatin affinity purification.
Methods Mol Biol. 2012;809:237-53
Authors: Harada R, Nepveu A
Abstract
Chromatin immunoprecipitation (ChIP) has become an essential assay in the field of transcriptional regulation. It is currently the most popular method to monitor the in vivo interaction between a protein and specific genomic sites. The method can also serve to identify novel transcriptional targets when the immunoprecipitated chromatin, sometimes called chipped DNA, is used either as a probe in hybridization experiments with microarrays of genomic DNA (ChIP-chip) or as template in DNA sequencing (ChIP-Seq). ChIP assays rely on the availability of good antibodies that can specifically and efficiently immunoprecipitate the protein under study even after cross-linking. However, good antibodies are not always available. To circumvent this problem, we have developed and validated the method of chromatin affinity purification (ChAP). The subsequent microarray analysis is then referred to as ChAP-chip. In brief, the protein under study is expressed together with two tags in order to allow the purification of chromatin by tandem affinity purification. To ensure that only true targets are identified, it is important to express the recombinant tagged-protein at physiological level. This requirement is not trivial as most expression vectors are designed to express proteins at high levels. We found most convenient to use an inducible retroviral vector in the absence of inducer and transactivator protein. We describe the procedure to generate cells stably expressing recombinant tagged-proteins at physiological level and then to purify the associated chromatin by affinity purification. Targets identified in this manner were validated in independent ChAP assays as well as in ChIP assays using antibodies against the endogenous protein.
PMID: 22113280 [PubMed - in process]
More...