How to sonicate chromatin: Sonication ChIP Protocol | CST Tech Tips

Chromatin fragmentation is a crucial step in the ChIP Sonication protocol. Here are some tips to improve your results.
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Transcript:
- How do I sonicate my chromatin in the sonication ChIP protocol? I am Fang Chen, Senior Scientist in the ChIP group at Cell Signaling Technology, and this is CST Tech Tips.
CST has two ChIP protocols, one using sonication, and one using enzymatic methods for chromatin fragmentation. This video covers the sonication protocol, in which sonication facilitates both cell and nuclear lysis, and fragmentation of the chromatin. If you are performing ChIP with the enzymatic protocol, refer to our other video of sonicating in the enzymatic ChIP protocol.
Whichever protocol you are using, always keep sample on ice during and between sonication cycles to avoid warming up. Second, you want to insert the sonicator probe tip to the bottom of the tube and adjust sonicator settings, like amplitude, to avoid bubbles during sonication. Otherwise, your target protein could get denatured and you can lose your ChIP signal.
For ChIP and ChIP-seq, the ideal fragment size is less than 1000 base pairs. In the sonication protocol, chromatin fragment size depends on total sonication time. As you can see in this sonication time course experiment, sonication for four, eight, or 12 minutes all yield fragmented chromatin DNA of the desired size, and longer sonication time leads to slightly smaller chromatin fragments. However, when we look at the target enrichment using these chromatin samples, 4 minutes of sonication provides the optimal level of target enrichment.
More interestingly, you can see that over-sonication is even worse than under-sonication. Over-sonication likely damages the chromatin integrity by either dissociating the target protein from DNA, or by denaturing the target protein or its antibody epitopes. Therefore, we suggest you spend a little time optimizing your assay to identify the minimal amount of the sonication required to obtain the desired size of chromatin fragments.
Thank you for watching. You can find full protocols for all applications on each antibodies specific product pages at cellsignal.com. If you have any questions about an antibody or protocol, you can get in touch with one of our scientists at cellsignal.com/support.
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👉About CST: Cell Signaling Technology (CST) is a private, family-owned company, founded by scientists and dedicated to providing high-quality research tools to the biomedical research community. Our employees operate worldwide from our U.S. headquarters in Massachusetts, and our offices in the Netherlands, China, and Japan. cellsignal.com/about
#CSTTechTips #ChromatinImmunoprecipitation #ChromatinIP #Sonication #Antibody

[@jamescarmody4713]

Thank you for the tips. I think it would be nice to show a demonstration of proper technique as well as showing data gathered.

[@jayvadanvaishanav]

what amplitude was used in this protocol?

[@cellsignaldotcom]

Hi, please refer to Section III, Step 5 of the Sonication protocol: www.cellsignal.com/learn-and-support/protocols/protocol-simplechip-plus-sonication. If you have further questions, please visit www.cellsignal.com/learn-and-support/resources-tech-chip and/or cellsignal.com/support.

[@user-sw8zq6ss1t]

Absolutely useless video with a clickbait name. No any details about amplitude, phoresis protocol or Sonicator model. Absolutely nothing.

[@cellsignaldotcom]

As mentioned in the video, the optimal sonication settings varies by sonicator, and it can also vary by sample type and fixation method.That's why we don't provide one set of sonicator settings in the video, instead we recommend optimizing. Please refer to Appendix C of the protocol: www.cellsignal.com/learn-and-support/protocols/protocol-simplechip-plus-sonication.
Please also feel free to contact our techincal support if you have further questions: www.cellsignal.com/support