Part one of this series described the importance of including proper controls to your protocols. In Part 2 we discussed how chromatin preparation affects the final outcome of the experiment. Now, let's take a minute to consider the immunoprecipitating antibody.
Antibodies that are not highly specific to the target of interest may bind unpredictably and increase the background noise, and this may make it more difficult to detect less abundant or lower stability interactions.
Your antibody should meet the following criteria.
It should be target-specific:
It should have an acceptable signal-to-background ratio:
The quality of the antibody isn’t the only factor that determines the outcome of the immunoprecipitation; the concentration of antibody can also dramatically affect your results. If the antibody concentration is too high, relative to the amount of chromatin, it may saturate the assay, leading to lower specific signal and/or increased background noise. Conversely, if the concentration of the antibody is too low it may fail to bind all of the target protein in the IP sample, resulting in less efficient immunoprecipitation.
ChIP-validated antibodies from CST are tested according to the criteria described above and each antibody is provided with a recommended starting concentration. But, if your antibody comes from another source you can use these guidelines to help ensure your antibody will work the way you expect it to.
Once the DNA is immunoprecipitated, it can be purified and you can move on to analyzing the results. That will be the focus of our next post, so until next time...
Read Part 4 in the series here: 4 Steps to Better ChIP results - Step 4 - Downstream Analysis
Check out our additional resources for chromatin profiling:
This is the third installment of a four-part series on how to improve your ChIP protocol. These posts are adapted from our full-length Guide to Successful Chromatin IP, which you can download by clicking on the button below.