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
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 during the 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
Sonication is a much harsher method,
which uses mechanical force to shear the DNA.
Sonication typically requires higher concentrations
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
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
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.