Chromatin Immunoprecipitation (ChIP) coupled with quantitative PCR can be used to investigate protein-DNA interaction at known genomic binding sites. if sites are not known, qPCR primers can also be designed against potential regulatory regions such as promoters. ChIP-qPCR is advantageous in studies that focus on specific genes and potential regulatory regions across different experimental conditions as the cost of performing real-time PCR is minimal. This technique is now used in a variety of life science disciplines including cellular differentiation, tumor suppressor gene silencing, and the effect of histone modifications on gene expression.
Workflow:
Chromatin preparation: cell fixation (cross-linking) of chromatin-bound proteins such as histones or transcription factors to DNA followed by cell lysis.Chromatin shearing: fragmentation of chromatin by sonication down to desired fragment size (100-500 bp)Chromatin IP: protein-DNA complex capture using specific ChIP-grade antibodies against the histone or transcription factor of interestDNA purification: chromatin reverse cross-linking and elution followed by purificationqPCR and analysis: using previously designed primers to amplify IP'd material at specific loci.
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