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What antibodies can be used in a CUT&RUN protocol? What controls should be included to measure signal-to-noise? These and other frequently asked questions are answered in this edition of CST Tech Tips.
● More answers to CUT&RUN frequently asked questions: cst-science.com/8qwn0e
● Watch the CUT&RUN animation video: • How CUT&RUN Profiles C...
● CUT&RUN benefits and reagents: cst-science.com/mdx241
● Get in touch with a CST scientist: cellsignal.com/support
Contents:
0:00 Intro
0:52 What antibody validation data should I expect for CUT&RUN?
1:43 What if I can’t find a CUT&RUN validated antibody for my target?
2:32 What controls should I use for my CUT&RUN experiment?
4:11 What do I do if my beads are clumping together?
4:47 What do I do if there isn’t enough DNA to quantitate before library construction and NGS?
5:38 Should I use phenol-chloroform extraction or spin columns to purify the DNA from CUT&RUN?
Partial transcript:
Today, we’re going to highlight the top five frequently asked CUT&RUN questions. I’m Fang Chen, senior scientist at Cell Signaling Technology, and this is CST Tech Tips.
CUT&RUN is a low cell alternative to chromatin immunoprecipitation or ChIP assays, used to profile protein-DNA interactions. Since CUT&RUN is a newer technique, you may have some questions related to the protocol or troubleshooting. We’re happy to provide some answers to help you ensure your experiments will generate the best possible data.
First, what validation data should I look for an antibody to support its specificity and sensitivity in CUT&RUN? Look for qPCR and NGS validation data, performed specifically with a CUT&RUN protocol including controls. For qPCR analysis, compare signal at positive loci in the genome, where the protein is known to bind, relative to negative loci, and compare the antibody to an isotype control to evaluate its specificity. The signal relative to input can give you an idea of the antibody’s sensitivity. For NGS, check that the provided data supports enrichment by the antibody at large number of peaks with a high signal to noise ratio,and enrichment of DNA at known target genes or loci.
If you don’t see an antibody for your desired target with CUT&RUN validation, consider antibodies that have been validated for ChIP-seq as a first option, or for ChIP as a second option. You can also consider antibodies that have been validated for immunofluorescence, abbreviated IF on the cellsignal.com website. A paper by Skene and Henikoff suggests some antibodies that fail in ChIP but work for IF might be expected to perform in CUT&RUN, since binding conditions are similar in the latter two assays.
However, not every ChIP or IF validated antibody will work for CUT&RUN. If you decide to try an antibody that has not yet been validated for CUT&RUN, you should definitely run it through your own validation testing including controls, which brings us to our next question:
What controls should I use for my CUT&RUN experiment? We recommend that you include both positive and negative controls to confirm your protocol is set up correctly, and to help troubleshoot if you don’t get the expected results.
Start by including a CUT&RUN-validated, positive control antibody to a target known to be bound to chromatin, such as this tri-methyl Histone H3 lysine 4 antibody, and a negative control antibody such as this IgG isotype control, and compare the signal for your target to both controls.
For qPCR analysis, select a primer set for a region of the genome where your protein does not bind, as an additional negative control. Check signal strength and signal-to-noise ratio for the positive antibody to confirm the assay is working well in your hands. This signal-to-noise is calculated by dividing signal from the positive primer set by negative primer set. Next, you can calculate signal-to-noise for your target antibody, again by dividing positive and negative primer sets. Then, calculate the ratio of the target antibody to isotype control, measured at the same primer set. All of these ratios should be three to one, or higher. For NGS analysis, include an input DNA control for signal-to-noise measurement. Input DNA is more diverse, and has lower background in NGS, compared to an IgG control.
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
Cell Signaling Technology and CST are registered trademarks of Cell Signaling Technology. All other trademarks are the property of their respective owners. cellsignal.com/trademarks
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17 июл 2024
