Performing a successful ChIP assay

In order to guarantee the success of a ChIP-on-chip experiment it is important to optimize the conditions for the equivalent single-ChIP experiment. Many protocols describing ChIP assays (see a schematic diagram in Figure 14.2) have been published and are now easily accessible.

There are two major considerations when setting up an experiment: the proper fixation of DNA-protein contacts, and the fragmentation of chro-matin. It is also very important to ensure that the antibody is highly specific and able to immunoprecipitate.

The most common crosslinking agent used in ChIP analysis is formaldehyde, a dipolar reagent that produces both protein-nucleic acid and protein-protein crosslinks, through the imino group of amino acids, such as Lys, Arg and His, and DNA (adenines and cytosines). A key property of

ChIP assay

Critical steps


1% HCHO, 15-60 min


300-1000 bp fragments


Ensure antibody specificity

Crosslinking reversal

65°C, 4-6 h

Figure 14.2.

Schematic representation of the ChIP assays. The central column shows the critical steps in the ChIP assay, as discussed in the text, and several technical tips are indicated in the right column.

the crosslinks obtained by using formaldehyde is their reversibility, which is achieved by treatment at low pH in aqueous solution or incubation at 60-70°C in the presence of SDS. Due to the small size of formaldehyde (2 A) only proteins located within this distance of the DNA will become crosslinked. Some of the chromatin-modifying enzymes, such as histone deacetylase, do not directly bind DNA and their gene-specific regulatory functions occur through recruitment by additional DNA-binding proteins that associate regulatory sequences. Although these proteins exhibit no DNA-binding properties, it is possible to investigate their association with particular sequences by using additional protein-protein crosslinkers (16). For instance, dimethyl adipimidate (DMA) has been used to investigate the association with the yeast HDAC Rpd3 (17).

Efficient fixation of proteins to DNA is crucial for the ChIP assay. Standard conditions for formaldehyde crosslinking usually consist of a concentration of 1% and incubation times between 15 min and 1 h, depending on the proteins to be analyzed. It is important to avoid a long formaldehyde crosslinking treatment as this increases resistance to fragmentation by sonication and decreases the efficiency of the technique. Moreover, formaldehyde is a moderately denaturing agent for proteins and a high concentration or long exposure to this reagent may result in the loss of antigen epitopes. It is advisable to determine empirically the effects of formaldehyde on the protein under study. After standard fixing conditions for different exposure times, immunolocalization analysis can detect loss of fluorescence signal due to denaturation.

When choosing fixation conditions, it is important to ensure that the increased mechanical resistance of chromatin still allows fragmentation by sonication. In fact, the size of the chromatin fragments is the second critical consideration when performing ChIP assays, since these will determine both the yield of immunoprecipitated material and the degree of resolution of the technique. Chromatin fragmentation is generally achieved by sonication (although micrococcal nuclease can also be used in protocols that avoid fixation by formaldehyde) and conditions must be optimized for each sonicator prior to any immunoprecipitation experiment.

In many studies, accurate mapping can be achieved by designing primers that amplify DNA fragments of 200-300 bp. Large chromatin fragments are specifically immunoprecipitated less efficiently than small fragments. Nevertheless, the size of the fragments determines the resolution of the technique and, therefore, fragments should not greatly exceed the size of the sequence to be analyzed. If the average chromatin fragments are much larger than the sequence to be PCR-amplified or probed, one cannot be sure that the protein for which the antibody was used is actually bound to that particular region or to a neighboring region.

Finally, the quality of the antibody is extremely important in ChIP assays. It is essential to ensure, firstly, that the antibody efficiently recognizes the antigen and, secondly, that most of the immunoprecipitated material represents specific DNA sequences. Ideally, a 'no-antibody' control and pre-immune serum control should be included.

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