4.4 ChIP Chop
Specific immunoprecipitation of methylated DNA itself has proved problematic; the available antibodies against 5-methyl-cytosine do not seem to immunoprecipitate methylated DNA. To overcome this problem, post-ChIP hydrolysis (‘chopping’) of DNA that is methylated at multiple purine-cytosine sites is performed using an enzyme called McrBC. Fragmentation within the target sequence prevents PCR amplification. With this technique it is possible to determine whether the DNA associated with a particular antigen is heavily methylated or not (Lawrence, 2004).
5. Quantification
Although hybridization using labelled probe sequences is still used to detect targets in the precipitated chromatin, PCR based methods are now more commonly used. Standard PCR can be used, but Real Time PCR (RT-PCR) is emerging as the method of choice due to its demonstrably quantitative nature, inbuilt controls for multiple product generation, and compatibility with TaqMan® and other stringent probe technologies. A typical set-up might use TaqMan® probe and/or Sybr Green technologies® to detect reactions carried out in an RT-PCR instrument such as the ABI 7000 according to the manufacturer’s protocols.
6. Analysis
Enrichment of a target is not solely dependent on the quantity of antigen associated with it. Precipitation will be affected by the accessibility of that antigen in that particular chromatin environment, the affinity of the antibody and the precise conditions of the immunoprecipitation. For this reason, the level of enrichment is always expressed as a ratio of bound (or precipitated) sequence over input. This also means that absolute levels of different antigens present at the same sequence cannot be directly compared. An example of an actual ChIP experiment is shown in figure 3.
Figure 3: Example of a result from a ChIP experiment
7. Protocols
A large number of protocols for ChIP have been published. We have included protocols from well respected researchers for chromatin preparation, NChIP and XChIP within the Abcam Chromatin and Gene Regulation webpage.
References:
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