Antibody Selection for ChIP


How to Select the Best Antibody for ChIP assays
Chromatin immunoprecipitation (ChIP) is a powerful technique for assessing what site of DNA to which a protein binds. This method has led to the characterization of many different interactions. Despite its power, ChIP runs into several significant issues. Many are related to the optimization of sample preparation that lead to no signal or high background. Perhaps the most important measure that needs to be controlled carefully is the selection of an antibody. It needs to meet several criteria before you can be sure it is useful.

It must be selective. The antibody you choose needs to recognize your protein of interest alone. Once the assay is performed, you will not have a way to which protein the DNA stuck. Typically, researchers will take for granted that the antibody only purified the protein of interest, but what if it also recognized a different DNA binding factor? The solution to this problem is to run as many assays as possible to assess selectivity of the antibody. The first and arguably the most important is a western blot. If you detect only one band out of a cell lysate, it is typically safe to assume that the antibody is reasonably selective.

It must recognize the protein when it is folded. Remember, you are purifying a protein from its natural state. The immunoblot you used to assess selectivity of the antibody may not tell you the whole story. Proteins immobilized on nitrocellulose have been denatured and reduced. They have significant folding, and therefore the antibody is designed to look for specific sequences of amino acids. In the native state, the part of the protein that is being detected might be buried within the folded structure. How can you control for this phenomenon? You may consider running the protein itself on a native gel and run a western blot on the full length protein.

In general, the best path to follow is doing your homework about the antibody of interest. Oftentimes other researchers have used the same antibody to perform different assays. You can usually trust consensus to find a place to start. Once you have obtained an antibody, make sure to run as much quality control as possible. Otherwise, you may find yourself moving down a path of bad results. You may also need to test several antibodies to assess which gives you the best signal. For the best results, you must empirically determine the best conditions.