Looking for the original paper that first describes the ChIP method was not an easy task: Most labs often cite their own adaptations of the technique, others simply quote to Alexander Varshavsky, who was the pioneer in the studies of histones using chromatin fixation; and yet other people just make reference to the original development of formaldehyde fixation of DNA and proteins. Here is a paper by Orlando et al. (1997) of what it could be considered the very first original description of the modern ChIP as we know it now.
Chemical crosslinking of cytosine and lysine by formaldehyde. In reaction I, a Schiff base is formed between the nucleotide and HCHO, and then this intermediate can react with a second amino group (from lysine in this case) and a condensation occurs (From Orlando et al., 1997).Taking the explanation of the authors and as it was mentioned in the above paragraph, this method is based on the formaldehyde fixation, that could cross-link proteins bound to nucleic acids or protein complexes in a reversible reaction (but does not react on dsDNA alone), with a very good resolution (range 2A). Cells are first fixed between 5 min - 1 hr, by addition of this reagent directly to the growth medium, then chromatin is solubilized by mechanical shearing (tipically sonication), because fixed cells are very resistant to nuclease treatment. Careful standardization of this sonication step should be carried out before further processing: variables such as time, immersion depth and others need to be precisely adjusted. In this original paper, CsCl- isopycnic centrifugation is employed to purify the chromatin; in the present times, after immuno- precipitation with monoclonal antibodies, DNA is extracted using silica- based commercial kits, or using first RNase - Proteinase K treatments followed by organic purification, and later a PCR amplification is performed to identify the sequence captured with the joined protein. Also, Orlando et al. (1997) use a Southern hybridization analysis, a step that is not carried out anymore, unless especially needed. As expected, the sensitivity of this method relies mostly on the quality and specificity of the monoclonal antibodies used; also the sonication step is determinant. We have already discussed here other ChIP adaptations such as ChIP-on-chip on a previous post ("High-throughput methods of regulatory element discovery") and there is also a good review of the ChIP method (Trends Biochem Sci 25:99 - 104) by the author himself.
Description of the steps in the original ChIP development; the Southern analysis has been intentionally ommited (Adapted from Orlando, 2000).
Orlando V., et al. 1997. Methods 11: 205-214 
Description of the steps in the original ChIP development; the Southern analysis has been intentionally ommited (Adapted from Orlando, 2000).
epigenetics
