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 these criteria:
It should be target specific:
- The antibody should demonstrate expected expression in positive/negative control cell lines, knockout cells, or siRNA-treated cells.
- The antibody should show appropriate expression in response to enzyme-specific activators and/or inhibitors
- The specificity of modification-specific histone antibodies should be verified using a peptide array or peptide ELISA.
It should have an acceptable signal-to-background ratio:
- Signal to noise should be assessed using isotype controls.
- The enrichment of known target genes should be at least 10-fold above background, as determined by real-time PCR analysis
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...
Part 4 . . . now available here
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.