Troubleshooting ChIP


As with any multi-step molecular biology method, chromatin immunoprecipitation (ChIP) can show you some strange results and problems at times. You need to understand the many ways in which it can go wrong. This will allow you to properly troubleshoot the method. Here is a list of some of the common problems and how to address them.

High Background That Is Also Found in Controls
If you are experiencing high background, non-specific binding to beads is often the first culprit you should exclude. The matrices on which the proteins are conjugated tend to be hydrophobic and attractive to an array of different molecules. You can eliminate this problem by pre-clearing. Pre-clearing refers to adding the lysate to beads for some time to get rid of the non-specific binding components. Then you can remove the beads and proceed with the addition of antibody.

Another cause of high background is contamination of your samples. Make sure you follow clean procedures at all times when dealing with DNA methods.

Poor Resolution Coupled to High Background
If you experience this issue, then your fragment of DNA is likely too big to resolve properly. You will need to adjust your sonication conditions to achieve smaller fragment sizes. Consider using enzymatic digestion of DNA to get small fragments instead of sonication. Enzymes can give you much smaller pieces.

Poor Signal
This is probably the worst offender. It is probably going to be the issue you face the most often. The reason is that a signal that is too low can be caused by many different problems. First, the piece of DNA bound to the protein might simply be too small. In this case sufficient detection will be very difficult to achieve. Excessive crosslinking is another common culprit for lost signal. Overexposure to formaldehyde can promote the masking of epitopes. This will prevent the protein from being properly immunoprecipitated. Without immunoprecipitation, you lose the DNA of interest. This can be especially problematic if you use monoclonal antibodies that have a very limited range of epitope selectivity. This is great for getting specific binding, but it can cost you if you begin masking the epitopes with too much fixation.

You may find that you did not add enough reagent to different steps. The amount of chromatin DNA you start with needs to be sufficiently high to purify significant amounts by immunoprecipitation. In addition, you must have the right amount of antibody to pull enough of the DNA-bound protein out of solution. Other issues with the antibodies include poor binding conditions. Too much salt or an altered pH can generate differences in the binding capacity of an antibody, so make sure your buffer is carefully made. Make sure you use the right kind of antibody binding beads. It is easy to mix up protein A and protein G.

Poor or problematic Amplification by PCR
PCR can almost feel like an art unto itself. If you accidentally contaminated your PCR reaction with outside DNA, you will see high signal even in your negative control. In this case, you will just need to start over. If you do not get any amplification, make sure to include a set of positive controls that will ensure your primers are in proper working condition.