Protein tag?

Question

I need to tag 2 proteins to do a co-immunoprecipitation. Do I tag one protein at N-terminal and the other at C-terminal or it does not matter which end I have the tag?

Thanks!!!

Answer 1

It can, but it just depends on the protein.  If the business end is really close to the N or C terminal, you can disrupt protein function, or an interaction.  If you can get both constructs (that is N- and C-terminus) easily enough, then go ahead and try.  If you can't, the only problem would be with a negative result.  If it does interact and pull something down, then you should be fine.  If it does not, and there are known interaction sites, or important structural determinants near the tag, then you would want to try the tag on the other side.

Answer 2

It doesn't matter which end, in general.  I've done studies like that with an N-terminal FLAG tag on one protein and an N-terminal myc tag on the other.

It helps a bit if you know where the proteins interact, and in theory either end could be inaccessible to antibody in the 2-protein complex, but you never know until you try.  It sounds like something you won't actually know until you've done these experiments and then mapped binding sites.  It's just not typical for the extreme N- or C-terminus to be part of a binding site, and thus not typical for your choice of tag site to matter.  I've been tagging the N terminus out of habit for 20 years and never had a problem.

But it's not unheard-of. I work with some proteins that bind to the C-terminal peptides of other proteins, so N-terminal tags would be important on those other proteins and C-terminal tags would interfere with binding, but that's an unusual situation.