So your experiments and data are funneling you down an inescapable path. You need to show direct gene regulation by your protein of interest. You think to yourself, “Oh, ChIP...”
The success or failure of a chromatin immunprecipitation (ChIP) experiment is highly dependent on the integrity of both the chromatin and the protein epitope, so your method of chromatin preparation can have a significant impact on the quality of your results.
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.
Part one of this series described the importance of including proper controls to your protocols. Now, we will examine how chromatin preparation affects the final outcome of the experiment.
First consider the type of interaction you are trying to detect:
- High-frequency, very stable protein-DNA interactions like those between histones and DNA, occur frequently enough that they may still be detected even if the protocol is not fully optimized.
- Low-frequency, less stable interactions like the binding of polycomb group proteins to specific genes (e.g., Ezh2), may fall under the detection threshold if the protocol fails to safeguard the integrity of the protein and the DNA, or if it relies on an antibody that is not highly specific to the target of interest.
Now it's time to consider your options for preparing your chromatin...
You're a confocal maestro, playing the lasers and the filters and the gain settings like a Stradivarius to generate those eye-catching, data-filled images that turn your peers green with envy. You sit back, work a cross-word puzzle, and watch with confidence while the red and green lights dance down your screen. But what happens when you reach the slide with that random treatment your advisor insisted you include (over your sound objections) . . . and you find your favorite cytoplasmic protein homing into the nucleus like the graduate students after free pizza?
You'll make a bee-line for your advisor, thinking "I may have to eat a little crow, but it's totally worth it." And, right as you're about to knock on his/her door it will hit you - oh, no! - I'm going to have to figure out how to do ChIP.
Don't worry - we can help!
Chromatin Immunoprecipitation (ChIP) is used to examine interactions between protein and DNA within the natural chromatin context of the nucleus. ChIP experiments first require fixing the cells, which cross-links the protein-DNA interactions into place. The chromatin is then broken into fragments and an antibody is used to immunoprecipitate the protein of interest along with any bound DNA. Finally, the cross-linking is reversed and the precipitated DNA is purified. The purified DNA can be subjected to further analysis, such as standard or real-time PCR, microarray, or sequencing.
These experiments are sensitive to the integrity of the chromatin, the quality of the protein epitope and the specificity of the immunoprecipitating antibody; and these variables become even more critical when the protein-DNA interaction under investigation occurs rarely or is unstable.