How to Select an Antibody for Protein Immunoprecipitation (IP) | CST Tech Tips

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Enrichment of proteins by immunoprecipitation is used in a variety of applications and protocols. In this Tech Tip, Sarah goes over some of the basics of the immunoprecipitation technique, including:
▪︎ Native vs. denaturing IP protocols;
▪︎ What to consider when choosing antibodies for IP;
▪︎ Protein A vs. Protein G beads for use with 🐁 mouse vs. 🐇 rabbit IP antibodies.
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Transcript:
What is immunoprecipitation used for, and how do I choose antibodies to immunoprecipitate my protein? I’m Sarah, Senior Research Associate at Cell Signaling Technology, and this is CST Tech tips.
Immunoprecipitation, or IP, can be used to enrich your protein of interest prior to analysis. There are many variations of IP, but the basic concept is to bind the target protein with an antibody -which I’ll refer to as the IP antibody - and then precipitate the resulting immune complex on beads derivatized with proteins that bind to the antibody. Following IP, proteins could be analyzed by western blot, mass spectroscopy, or other assays. IP can aid detection of your protein if it is expressed at low abundance, or if you are working with a limited sample.
Do you already use IP in your experimental workflow, or are you planning your first IP experiments? Let us know in the comments section below this video! And while you’re there, give this video a like, and share it with your colleagues.
Okay, let’s consider the conditions in IP compared to western blot and how that affects binding of the IP antibody. In native IP, during and after cell lysis, proteins are kept in their native, folded conformation during the IP reaction.
Native IP is suitable for antibodies whose epitopes are on the surface of the protein. But some epitopes may be buried in the center of the protein structure, or located in a binding interface that becomes obscured when other proteins are bound to your target protein. An antibody that recognizes one of these epitopes might be able to detect your protein in the denaturing conditions of a western blot, but unable to bind its epitope and capture the protein in Native IP.
For these cases, an alternate protocol, using reducing conditions to partially denature the protein, may be beneficial. Before setting up your IP, check for validation data from the antibody supplier, and check whether a native or denaturing IP protocol is recommended for that antibody. The immunoprecipitated complex can be prepared for western blotting by washing with lysis buffer and then resuspending and boiling in SDS sample buffer.
When it comes to choosing what type of beads to use, the choice of Protein A versus Protein G beads should be based on the host species of the IP antibody. We recommend Protein A agarose or magnetic beads for IP with rabbit antibodies and Protein G agarose or magnetic beads with Mouse antibodies.
Magnetic beads have gained in popularity, and magnetic separation affords a faster protocol without the need for centrifugation. But whichever format you use, it’s always a good idea to incorporate proper controls.
Choosing antibodies for downstream western blot analysis involves other considerations for your experiment. We’ll cover this topic, as well as the different types of experimental controls you should use for IP, in future Tech Tip videos.
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[@seussdoctor9452]

Hello, thank you for the explanation. The part about tips on considering whether my antibody recognizes native or denatured protein was interesting.
Also, an introduction of how to choose lysis and elution buffers for Co-IP experiments would be most helpful.

[@cellsignaldotcom]

We have a video on lysis buffers here: ruclips.net/video/KV51wMtVNak/видео.html