Chromatin Immunoprecipitation and Quantitative Real-Time PCR to Assess Binding of a Protein of Interest to Identified Predicted Binding Sites Within a Promoter

Chromatin immunoprecipitation (ChIP) has become a widely used methodology for assessment of protein/DNA interactions. The technique allows the analysis of direct binding of transcription factors to gene promoters, identification of histone modifications, and localization of DNA modifying enzymes. Antibodies conjugated to agarose beads can be utilized to immunoprecipitate specific proteins, cross-linked to sheared chromatin regions to which they are bound endogenously. With downstream applications including quantitative real-time polymerase chain reaction (qRT-PCR), genome-wide sequencing (ChIP-seq), microarray analysis (ChIP-chip), and mass spectrometry (ChIP-MS), the technique enables comprehensive assessment of protein/DNA interactions. Here I describe ChIP, followed by qRT-PCR, to assess direct binding of a single protein to multiple predicted binding sites within a gene promoter.