The amount matters as well: the more cell lysate you use, the more of your protein you can pull down. Some things in science, like the best lysis buffer, just need to be determined empirically.ĭon’t forget to use protease inhibitors when lysing your cells to avoid your protein getting eaten up by proteases, which also get released during cell lysis. Set time aside to test a number of lysis conditions to find the optimal lysis solution. This kind of lysis is least likely to disturb any protein interactions.įor less soluble protein complexes, however, lysis buffers may need to contain non-ionic detergents such as NP-40 or Triton X-100. Non-detergent, low-salt lysis buffers are a popular choice for Co-IP of soluble proteins. The method of lysis is important in Co-IPs. Here you gently break open your cells to make your protein accessible to the antibody. The general steps are as follows (Figure 1).įigure 1. A Simple Co-immunoprecipitation ProtocolĬo-IP protocols are very similar to traditional IP protocols, with the difference that Co-IPs require more gentle assay conditions to maintain the interaction with binding partners. You can then go on to study binding kinetics using SPR techniques. Furthermore, you can use Co-IPs to determine protein–protein interactions under varying conditions. Thus, Co-IPs are a great way to identify protein complexes. When successful, Co-IP pulls down not only your protein-of-interest but also its interaction partners. However, a Co-IP requires greater care and more physiologically relevant conditions than traditional IP. Your protein-of-interest is “pulled down” and out of solution by an antibody, which is, in turn, captured using beads. With both techniques, you use antigen/antibody interactions to isolate proteins. In essence, Co-IP is just a more delicate version of a traditional immunoprecipitation, which can be thought of as a small-scale affinity purification. The Basics of Co-immunoprecipitation Experiments It will also touch on how you can use Co-IP with SPR applications to gain a detailed insight into how your protein interacts with others. This article will get you ready for your first Co-IP, provide a handy Co-IP protocol, and discuss Co-IP controls you should include. Do you wonder if your favorite protein interacts with another protein? Do you wish that you could shine a spotlight on your protein to determine its binding partner? You can use co-immunoprecipitation (Co-IP) to find your protein’s partner.
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