Perhaps you’ve never done ChIP before and need to establish the technique in your lab, or maybe you use ChIP routinely and need further tips to improve your results? Read on and discover four steps to take your ChIP expertise to the next level:
- Cross-linking or not? The aim of cross-linking is to fix the antigen of interest to its chromatin binding site, so the further away the interaction of interest lies, the less effective ChIP will be without cross-linking. ChIP for histone modifications is unlikely to require cross-linking whereas nonhistone proteins such as transcription factors and proteins contained within DNA binding complexes will probably need cross-linking. If cross-linking is used, ChIP reaction will be known as X-ChIP, whereas a native ChIP reaction will be known as N-ChIP.
- Be careful when fragmenting your chromatin. Fragmentation of the chromatin is required to make interactions accessible to antibody reagents. Whatever method you are using be sure to run a fragmentation time course every time you set up an experiment. For X-ChIP sonication is necessary as formaldehyde cross-linking restricts the access of enzymes to their targets. Sonication creates random DNA fragments (average of 500–700 bp). When doing N-ChIP, enzymatic digestion with micrococcal nuclease should be able to generate single monosomes (~175bp). Keep in mind that micrococcal nuclease favors certain genomic sequences over others and will not digest DNA evenly.
- Make sure the antibody works in ChIP. If available, use an antibody that has been fully characterized and labeled as ChIP-grade. If there is none available, then an antibody that works in normal immunoprecipitation or in applications that require recognition of epitopes in their native forms is a good candidate. A polyclonal antibody is generally preferable to a monoclonal; monoclonal antibodies recognize only a single epitope, which could be masked during the cross-linking process. On the other hand monoclonal antibodies have the advantage of greater batch-to-batch consistency.
- Use the right controls. As a positive antibody control for the technique, histone H3 tri-methyl K4 (H3K4me3) and tri-methyl K9 (H3K9me3) are popular positive controls to use when chipping active and inactive genes respectively. As a negative control, use an antibody that recognizes a nonchromatin epitope such as anti-GFP antibody. Remember though that these antibodies are not positive and negative controls per se, as this will depend on the locus you are studying. If there is no H3K4me3 at the particular locus of interest, the best anti-H3K4me3 ChIP-grade antibody in the world will not immunoprecipitate anything from this region and therefore will not be an appropriate positive control. If ChIP is not an established technique in your lab, consider using a kit.