Transcription Factor Targeted ChIP-seq Kit

Background

Association between proteins and DNA is a major mechanism in many vital cellular functions such as gene transcription and epigenetic silencing. It is crucial to understand these interactions and the mechanisms by which they control and guide gene regulation pathways and cellular proliferation. Chromatin immunoprecipitation (ChIP) is a technique to analyze the association of proteins with specific genomic regions in intact cells. ChIP can be used to study changes in epigenetic signatures, chromatin remodelling and transcription regulator recruitment to specific genomic sites. In ChIP, living cells are first fixed with a reversible cross-linking agent to stabilize protein-DNA interactions. The most widely used reagent to fix cells is formaldehyde which generates covalent bonds between amino or imino groups of proteins and nucleic acids. Formaldehyde treatment crosslinks both DNA-protein as well as protein-protein complexes. Following cross-linking, chromatin needs to be sheared very efficiently into homogeneous small fragments that can subsequently be used in immunoprecipitation (IP). After fragmentation, the sheared chromatin is precipitated with a specific antibody (AB) directed against the protein of interest. The chromatin-AB complex is isolated using magnetic beads. Finally, the precipitated DNA fragments are released from the AB, and analyzed. Enrichment of specific sequences in the precipitated (IP’d) DNA indicates that these sequences were associated with the protein of interest in vivo. Analysis of specific regions can be performed by quantitative polymerase chain reaction (qPCR). In recent years, ChIP combined with high-throughput Next-Generation sequencing (ChIP-seq) has become the gold standard for whole-genome mapping of protein-DNA interactions.