Chromatin Immunoprecipitation (ChIP)

Chromatin immunoprecipitation (ChIP) is often used to identify genomic regions associated with DNA-binding proteins, including histones, transcription factors, protein-gene interaction and loci, or can be secondarily used to study RNA-protein interaction. Because the proteins are captured at the sites of their binding with DNA, ChIP can detect DNA-protein interactions that take place in living cells.

Experimentally, ChIP usually involves cross-linking of the chromatin-bound proteins by formaldehyde. Sample then undergo sonication or nuclease treatment to obtain small DNA fragments. Traditional IP techniques are then carried out using specific antibodies to the DNA-binding protein of interest. After, DNA is released from the proteins and isolated DNA can be analyzed by PCR techniques, microarrays, or sequencing, Southern blot, or WB. There exist two main variations of ChIP: X-ChIP, and N-ChIP.

 

ChIP Variations

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X-ChIP

X-ChIp can be performed on all cell types, tissues, and organisms and is ideal for studying nonhistone proteins, DNA-protein, RNA-protein, and protein-protein interactions. It is more sensitive than N-ChIP, requires fewer cells, and less antibodies. X-ChIP uses formaldehyde fixation that lessens the possibility of protein and/or chromatin rearrangement.

Some downsides to this method lie in that excessive cross-linking can lead to difficulties in DNA fragmentation, which lessens DNA retrieval, necessitating post-ChIP amplification of the recoverable DNA. Formaldehyde fixation also has the potential to fix transient-DNA protein interactions, alter the binding properties of the antigens, and interfere with immunoreactivity. Additionally, enzymatic digestion is not possible after formaldehyde treatment, likely due to destruction of nuclease reactive sites.

N-ChIP

N-ChIP is typically used for native chromatin, unfixed, and nuclease digested samples. N-ChIP is suited for studying tightly bound proteins, like histones and their isoforms, but not for nonhistone proteins which may rearrange during processing. In N-ChIP, antigen binding specificity is higher than X-ChIP as antibody binding to unfixed proteins is stronger than to fixed proteins. DNA recovery is higher than in X-ChIP, so DNA amplification as a subsequent step is not necessary.

One limitation lies in that not all of the nuclease-digested chromatin will solubilize after processing, and some chromatin may remain in the nuclear pellet. High concentrations of nuclease may overdigest the chromatin, which can hinder the detection of protein-DNA interaction. Chances of chromatin rearrangement during processing are also highly likely.

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