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...
Step 2: Chromatin preparation –
Many labs rely on sonication to prepare their chromatin for immunoprecipitation. While effective, sonication requires exposing the chromatin to harsh, denaturing conditions (i.e., high heat and detergent) that can damage both antibody epitopes and the genomic DNA. In addition, sonication is inconsistent. The method and quality of the prep will vary depending on the type and brand of sonicator you are using and on the condition of the sonicator probe used. Additionally, there may only be a few-second difference between having chromatin that is under or over-processed. As a result, chromatin preparations of consistent fragment size are difficult to generate with this method.
In contrast, enzymatic digestion uses a micrococcal nuclease, which cuts the linker region between nucleosomes to gently fragment the chromatin into an array of uniform pieces. Enzymatic digestions do not require high heat or detergents and provide consistent results if the recommended enzyme to cell number ratio is used. Thus, enzymatic digestion is simple to control, protects antibody epitopes and DNA from shearing or denaturation, and results in a consistent, high-quality chromatin preparation that is conducive to immunoprecipitation.
The experiment shown below was performed using the SimpleChIP® Plus Enzymatic Chromatin IP Kit (Magnetic Beads) #9005 and another company’s sonication-based kit. Chromatin was first prepared using either enzymatic digestion (according to the SimpleChIP kit instructions) or sonication (according to the other company’s instructions). The chromatin was subjected to immunoprecipitation with the indicated panel of antibodies using immunoprecipitation reagents from either the SimpleChIP® kit or the other company’s kit. The immunoprecipitated DNA was quantified by real-time PCR and is presented as a percent of the total input chromatin.
The enzyme digested chromatin showed more robust enrichment of target DNA loci than sonicated chromatin, using either the other company’s IP kit or the SimpleChIP® kit. This was especially apparent when less stable interactions; such as the binding of polycomb group proteins (Ezh2 [D] or SUZ12 [E]) to specific genes were assayed.
Step 3: Immunoprecipitation – bringing down the DNA...available here . . .
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This is the second 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.
