CoIP: How to find if two proteins are acting sus with each other…
Co-immunoprecipitation (CoIP) is a proteomic method used to see if two proteins interact in vivo at a certain point in time. The first step is to lyse the cells of interest and incubate the lysate with antibody against one of the proteins. This antibody should be attached to a dense bead so that we can separate the antibody-protein complex. Then, wash all extraneous proteins away, so that you only have your bead and antibody bound to the protein.
At this stage, there are two possibilities. One possibility is that the two proteins interact, in which case some of the antibody-protein complexes will not just contain one protein, but two (if the proteins interact) - so the antibody will be bound to a 2-protein chain. The other possibility is that the proteins don't interact, so only one protein (the antibody-binding one) is the only one binding the antibody. Below is a diagram of this experiment:
This image depicts a case where the two proteins DO interact, so when protein X binds the antibody, Y is already bound to X so Y tags along too. If X and Y didn't interact, only X would bind the antibody as X and Y would be irrelevant to one another.
We can check this result using a Western blot, to see if only one or both proteins bound the antibody and thus see if they bind one another to assess interaction. In order to determine if the proteins interact, we need to see if the second protein is also bound to the first protein and the antibody-bead. We can thus use an antibody against the second protein (in the case of the diagram above, an antibody against protein Y) to assess if it is part of the complex. Because this is a Western blot, we will only see bands on the gel if the antibody bound protein Y, and we will have an empty gel if there is no interaction.
To recap:
1 - We used an antibody against protein X.
2 - We pulled down protein X and anything attached to it.
3 - We washed everything off, and just isolated the anti-X antibody, the X, and anything bound to the X (if any).
4 - We wanted to see if Y specifically was attached to X, so we added anti-Y antibodies to the mix. If these antibodies bound anything, we would see a band on the Western blot gel.
Yeah that's it. Thanks for reading, and sorry this one was short. It's 2am and things are kinda bad right now.
Here are some videos to help you learn more about this topic:
https://youtu.be/_LK9Q6_2d1k?si=B45GyTqNnhSQaCw1 - nice summary of CoIP process.
https://youtu.be/faOELRICYqY?si=Mi1lE6dSU44YTEH4 - summary of Western blot.
https://www.profacgen.com/Co-Immunoprecipitation-Co-IP.htm - nice article with some formal vocab like “bait” and “prey” - probably good to know. I was just trying to give a brief intro. But in short: X=bait, Y=prey.
As always, feel free to ask me any questions in the comments/DMs!
