3. ChIP experiments: factors for consideration
3.1 The antibody
For a successful ChIP experiment the choice of antibody is paramount. Typically, ChIP’ing antibodies recognise histones, histone modifications or chromatin-associated factors. The specificity of each antibody needs to be well characterized; antibodies should be tested in both ELISA and Western blot (using target and non-target antigens as competitors) to confirm specific epitope recognition. Western blotting can be used to demonstrate that the correct target has been successfully immunoprecipitated.
Antibodies that recognise multiple antigens in the chromatin fraction should be avoided (or at least this should be considered in the final interpretation). Immunofluorescence can also be used to check that antigen recognition occurs in a more natural context, and can also be combined with competition assays.
Perhaps the most stringent test of a ChIP’ing antibody is to perform parallel ChIPs in wild type and mutant backgrounds to demonstrate that the observed enrichment is due solely to the target antigen.
Finally, the quantity of antibody, and the conditions under which to use it, should be determined empirically for each antibody. Typical starting IP conditions would be 2-5µg of antibody per 50µg of DNA (where the concentration of DNA has been measured in a deproteinized aliquot of the chromatin fraction).
Figure 2: Chromatin Immunoprecipitation - the steps
