How to Optimize your Chromatin Fragmentation for ChIP
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The goal of this video is to guide you through chromatin fragmentation, a critical step in a Chromatin Immunoprecipitation (ChIP) experiment. It is important to consider whether the target protein is a histone modification, transcription factor, or transcription co-factor since each protein binds differently to DNA. Due to these binding differences, additional measures should be taken into account when optimizing chromatin fragmentation for each protein class, whether performing sonication or enzymatic digestion.
Chromatin immunoprecipitation, or ChIP, is a powerful research technique used to identify and analyze protein DNA interactions within the genome in vivo. This short video will focus on chromatin fragmentation, which many consider to be one of the most important steps in a ChIP experiment. It is critical that the cross-linked protein DNA interactions are preserved during chromatin fragmentation. Otherwise, the antibody against the protein of interest will not bind the chromatin fragment and pull it down duringthe immunoprecipitation.
Therefore, you need to consider whether your target protein is a histone, or a histone modification, a transcription factor, or a transcription co-factor, as each of these protein classes binds differently to DNA. Histones and histone modifications are easy to ChIP, because they are relatively abundant and bind directly to DNA in a very stable manner so they stand up well to harsh fragmentation conditions. Transcription factors are more difficult because they bind less stably to DNA, are less abundant, and show more sensitivity to harsh fragmentation methods. Finally, transcription co-factors are the most challenging protein class to ChIP because they often do not contact DNA directly, which makes cross-linking very inefficient. Co-factors are the most sensitive proteins to harsh fragmentation conditions. Understanding which type of target protein you are studying will help you choose the most appropriate chromatin fragmentation method. Either sonication or enzymatic digestion. Both methods are effective at breaking cross-link chromatin into smaller fragments, which is necessary to allow for efficient immune enrichment.
Enzymatic digestion uses micrococcal nuclease to gently digest the DNA under low detergent and low heat conditions. Sonication is a much harsher method, which uses mechanical force to shear the DNA. Sonication typically requires higher concentrations of detergents. And the mechanical shearing generates heat, resulting in harsh conditions that can denature chromatin proteins.
Since histones are resistant to harsh fragmentation conditions, both enzymatic digestion and traditional sonication can easily be used to generate a successful ChIP experiment, as shown here for trimethyl histone H3 lysine 27.
But which method is best for performing ChIP for transcription factors and co-factors? Enzymatic digestion works well to preserve transcription factor and co-factor binding and ensures efficient immuno-enrichment of these proteins.
Sonication also works, but some additional measures are necessary to ensure a successful experiment. First, using low detergent cell lysis and nuclear lysis buffers will help protect the chromatin integrity, resulting in increased immune-enrichment, making sonication more compatible for use with transcription factors and co-factors.
A second critical step is the optimization of sonication time or sonication cycles. Ideally, you want to use the minimum number of sonication cycles to achieve properly sized chromatin fragments and maximum chromatin integrity. More sonication is not always better. As you can see in this figure, the best results are achieved with as little as four minutes of sonication.
In summary, you should consider the following before starting your chromatin fragmentation. You want to have a clear understanding of the type of protein you are studying, and how strongly it binds to DNA. And if you plan on using sonication, whether the protein may be affected by harsh fragmentation conditions. If the protein is a transcription factor or co-factor, use either enzymatic digestion or sonication with a low detergent lysis buffers and minimal sonication time to generate properly sized chromatin fragments, and maximize enrichment of chromatin targets. To learn more about these methods, review FAQs, and troubleshooting tips for ChIP, click here.