I am having trouble understanding the different approaches where pull down vs co IP is used. I have to study protein- protein interactions associated with a specific protein. I have developed an over expression system where my target protein is tagged with myc- DDK tag. I have anti FLAG antibody as well as an anti-protein antibody (for my protein of interest) as well. I am confused as to which would be a better way to study the protein- protein interactions.
The anti-protein antibody is very precious and very costly!!!
Any suggestions regarding this?
I am not sure what you mean by pull down. In my understanding, the term "pull down" is just another way to refer to an immunoprecipitation (IP). Generally, I would call the procedure an IP and the action involved "pulling down" a protein rather than saying immunoprecipitating (this may be different in different lab cultures).
You can do a regular IP in which you pull down and analyze one target protein or a co-IP in which you pull down one protein, then look for the presence of another protein that was bound to it. Co-IP (vs. regular IP) is used for studying protein-protein interactions and is what you would want to do for your application.
When you do a pull-down there is no antibody involved. Instead of using an antibody against one of the proteins or a tag like you do in IP or co-IP, you use an affinity ligand coupled to a resin. You can for example use a His-tagged protein and do a pull down with a Ni-resin or pull down a GST-fusion protein using Glutathion as ligand.
Since your protein has a Myc and a Flag tag you can do a co-IP using an antibody against one of the tags or the protein itself. Since not all available antibodies work in IP you should then first test if the antibody that you want to use can immunoprecipitate your protein.
OH I've realized I've been calling them all IPs regardless of whether there is an antibody, which doesn't actually make sense. Yes, if your antibody doesn't work for IP or is too expensive, you can try an affinity resin for one of your tags. I don't know if there are any for the Myc or Flag tags that aren't antibody-based (which are usually pretty expensive themselves), but if it's really an issue, you can switch to a different tag.
In addition to the options mentioned in the previous comment, you can also do a Strep II tag with the Step Tactin Sepharose resin from IBA life sciences. It's pretty inexpensive for small-scale applications and works really well.
Either way (pull-down or IP) you can look for the presence of your interactor. The only thing that really changes about the procedure is your initial binding step.
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