3.2 The Chromatin
Chromatin from most sources, including tissue, is a suitable substrate for ChIP. While the isolation procedure necessarily varies depending on the source, two types of ChIP can be recognised: Native and Cross-linked, known as NChIP and XChIP respectively.
(i) NChIP: Native chromatin is used as substrate. The advantage of this procedure over XChIP is that antigens cannot be obscured or modified by the chemical cross-linking. However, this lack of cross-linking necessarily means that only proteins very tightly associated with the chromatin can be immunoprecipitated, typically limiting this type of ChIP to histones and their modifications (O’Neill and Turner, 2003).
(ii) XChIP: Cross-linked chromatin is used as substrate. XChIP is more widely used than NChIP since it allows analysis of a much broader range of chromatin-associated factors (through physical cross-linking of weakly associated proteins). However, some antigens may prove difficult to immunoprecipitate with XChIP; changes to the epitope or occlusion may occur in the cross-linking process. The use of rapid cross-linking has been successfully used to follow temporal changes in chromatin, such as histone modifications (Orlando, 2000). Cross-linking is typically achieved using formaldehyde or UV treatment.
After purification, the native or cross-linked chromatin is digested, typically by micrococcal endonuclease, or mechanically sheered by sonication to generate chromatin fragments. Empirical manipulation of either of these processes can be used to generate a range of fragment sizes. Although this fragmentation gives sufficient resolution for most experiments, fragments of a specific size can be further purified if required (e.g. by sucrose gradient centrifugation). In this way pure mononucleosomes can be analysed.
4. Flavours of ChIP
The conceptual limitations of ChIP analysis are only the antigens that can be targeted, and the ways in which associated DNA can be analysed. Novel techniques continue to be published.
4.1 Temporal ChIP
Chromatin can be analysed at multiple time points in response to specific signals. Although fixation times typically extend up to 15 minutes in duration, significant changes have recently been described between one-minute time points using this technique (Metivier et al., 2003).
4.2 ChIP on DNA micro-arrays (ChIP on chip)
Combining ChIP and micro-array technology allows genome-wide analysis of antigen distribution. Immunoprecipitated chromatin is quantitatively amplified and labelled using random primers, then used to probe an appropriate DNA microchip. In this way, given efficient precipitation and a comprehensive array, a complete genome localisation map for the protein or modification of interest can be assembled (Bernstein et al., 2002; Kurdistani et al., 2002).
4.3 Re-ChIP
As the name suggests two sequential immunoprecipitations are performed with two different antibodies. Successful re-ChIP identifies two targeted antigens as occurring together on the same chromatin fragments. This data may be used to assemble the proteins into higher order complexes. However, care must be taken not to over-interpret such information; it is possible to envisage two antigens occurring consistently in close proximity at a given sequence without existing in the same complex (Metivier et al., 2003).
