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Protein-protein interactions can be studied using co-immunoprecipitation (co-IP). In this Tech Tip, Sarah describes co-IP experimental design and the application of IP to study protein-nucleic acid interactions, and to proteomics.
• “Bait” and “prey” in co-IP experiments
• Application of IP techniques in ChIP, RIP, and proteomics
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Transcript:
- What does prey versus bait mean in co-IP experiments, and where else is immunoprecipitation used? I'm Sarah, Product Scientist at Cell Signaling Technology, and this is CST Tech tips.
OK, let's talk about some of the different variations of IP. If you're investigating protein-protein interactions, you can employ co-IP assays. The goal of co-IP is to enrich a protein complex consisting of one protein referred to as the bait, along with proteins called prey that are bound directly or indirectly to the bait.
Depending on what is known about protein interactions, what antibodies are available, and what questions you want to ask, you can use different co-IP strategies. In one strategy, your protein of interest acts the prey, and a known or suspected binding partner acts as the bait. In another strategy, the protein of interest acts as the bait. In both cases, you should select an antibody against the bait protein which has been validated for use in IP. Following co-IP, the prey protein can be identified by western blot or other analyses.
When designing your experiment, it's worth considering protein structure since binding of the IP antibody may be hindered by, or even potentially disrupt, the protein-protein interactions you're investigating. If you observe a weak band or no band in your blot, using an IP antibody to a different epitope on your protein of interest, if available, may improve signal.
When you do observe a positive result in your co-IP, try performing a reverse co-IP with the bait and prey proteins switched. This can give you additional confidence in your findings, although these results may be affected by cross-reactivity of the antibodies used.
Immunoprecipitation can also be used to study protein-DNA interactions in chromatin-immunoprecipitation or ChIP assays, or for protein-RNA interactions in RNA IP or RIP assays. The general principle is to IP with an antibody to a protein known or suspected to bind to DNA or RNA, respectively, and pull down the associated nucleic acid for analysis with sequencing or PCR. The protocol for ChIP is more involved than for protein co-IP, so check out our Tech Tip videos for ChIP if you'll be performing those assays.
Finally, IP techniques can be incorporated in proteomics studies. As just one example, you can enrich a population of signaling proteins carrying phosphorylation or other post translational modifications using specialized motif antibodies, and then identify those proteins using mass spectrometry. This enables investigations into how signaling activities are altered in disease states or by treatments with activators or inhibitors. To learn more, check out my CST colleague's presentation in the pop-up link in the corner.
Thanks for watching. If you found this video informative, don't forget to hit the like button and share it with your lab mates and subscribe for more Tech Tips. And any time you have a question about an antibody, or a protocol, you can get in touch with a CST scientist at cellsignal.com/support. Good luck with your experiments!
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