Learning Journal 4

A Technique

One technique that I have learned a lot about in BIOL 463 is Chromatin Immunoprecipitation, or ChIP. ChIP is an experimental technique used to investigate interactions between proteins and DNA in the cell. It allows the user to determine whether specific proteins are associated with specific regions of the genome. For example, ChIP can be used to analyse the binding of proteins such as transcription factors on promoters or other DNA binding sites. Another application of this technique is to determine the specific location of various histone modifications in the genome, therefore identifying the targets of histone modifiers.

Anything Difficult?

Emily, Sina and I had the great opportunity of teaching the ChIP technique to our classmates during the presentations of ‘commonly used’ experimental techniques. When we gave our presentations, there was one aspect of the technique that most students found confusing and it was also the same step that I found confusing when I was learning about ChIP. The experimental method that was quite difficult to understand was the use of a bead in the antibody-binding or immunoprecipitation step. The reason I believe this step was so confusing is because I, and most of my peers have probably only learned about antibodies directly binding antigens. The purpose of having a bead in the immunoprecipitation step was often presented unclearly. The antibodies are commonly coupled to agarose, sepharose or magnetic beads, which are then immunoprecipitated in complexes (i.e., the bead–antibody–protein–target DNA sequence complex). The bead provides a larger component whereby collection of antigen-antibody complexes can be made easier. The complexes are then washed to remove non-specifically bound chromatin, the protein–DNA cross-link is reversed and proteins are removed by digestion with proteinase K.

A Question to Test One’s Understanding

If I were to test whether someone truly understands how the technique above works, I would ask them what the purpose of each step is, and what would happen if this step was left out, or done incorrectly. In my experience, it can be fairly easy to memorize the sequence of steps that make up an experimental protocol. However, knowing the exact purpose of each step can show a thorough understanding of how the technique works. In addition, understanding what would occur if a step was excluded or done incorrectly would demonstrate knowledge of how that step relates to the rest of the experiment. For example, if the protein crosslinking never occurred, the student should say that proteins would never be fixed to the DNA they interact with. As a result, after immunoprecipitating, only naked DNA would be collected and we would not know which proteins interact with that DNA since they were always free to dissociate into the solution.