Researchers rely on high quality antibodies to perform successful chromatin immunoprecipitation (ChIP) experiments. When investigating the protein-DNA interaction of interest, valid and reliable results simply cannot be attained using nonspecific and inefficient antibodies. If the antibody doesn’t come “ChIP-qualified” or “ChIP-grade” from the supplier, there are several tips you can follow to determine if it’s likely to perform well in ChIP and won’t pull down distracting material to get in the way of a successful study.
Carry out the standard steps as you would if you were performing a chromatin IP protocol. It may be your best bet to use an antibody validated to a target for use in similar applications like immunoprecipitation, immunohistochemistry, or immunocytochemistry; if the antibody is not successful in these, it will most likely not perform well in ChIP.
ChIP demands antibodies to be more “elite” in their quality and fully characterized, so, success in a different application does not necessarily guarantee success in a more involved one, such as ChIP. As a preliminary measure you may wish to carry out Western blot, peptide dot blot, or ELISA on an assortment of antibodies and then test only those high in specificity.
Using a polyclonal antibody will help reduce the chance of masked epitopes during the cross-linking process, but monoclonal antibodies present less variation between batches and are often quite specific because they recognize only a single epitope. Ideally, going with an affinity purified antibody will increase your chances of success and minimize cross-reactivity.
Be smart about your controls. If possible, when choosing a positive and negative control locus, be sure to use a tested gene for which the modification or protein of interest has been established and one for which the literature supports the decrease or absence of your protein/modification, respectively. Conduct a real-time PCR to test these primers. Also, in order to catch possible contamination during your actual ChIP experiment, always use a no template control.
To improve signal strength and minimize background, try testing out a range of antibody concentrations as you carry out ChIP protocol. 1-10 ug of the antibody for each IP is considered a typical amount. Also, adjusting the stringency of the last wash can help account for the possibility that your antibody may have a low affinity for your target and strengthen the signal.
By following these tips you can narrow down whether or not an antibody is likely to perform well in a process that has been gaining popularity and refinement among researchers since the 1980s. Chromatin immunoprecipitation is an excellent method that, with the use of a high quality ChIP-validated antibody, can help researchers unlock the mystery of their protein-DNA interaction of interest.
