Hi guys, I am trying to detect the binding of 2 proteins by Co-immunoprecipitation. However, the choice of antibody is confusing me.
I tried probing the endogenous protein right away but the results is not good. Now I co-transfected the 2 proteins in HeLa, one carries a myc tag and another carries a flag tag. I want to capture the flag-protein on beads and detect the myc-protein on western blot.
For the choice of antibody, is it a must for me to use anti-flag antibody? Is it okay If I use antibody which target the protein instead of the recombinant tag for protein complex capturing?
On western blot, I know it is best to use antibodies with different species origin with the one I use for protein complex capturing but the same question applies : is it okay If I am using antibody that target the protein instead of the tag?
I am asking this question because I read a lot of publications. And it seems to be that when people co-transfected the cells before doing the Co-IP experiments, they often use antibody that target the recombinant tag instead of the protein. I wonder if it is okay if I am not doing in this way.
Thanks.
yes, you absolutely can do it. Because the antibody to a specific protein is expensive and sometimes is not commercially available, people choose anti-tag antibodies.
No you can use either you anti-flag/anti-myc or the antibody against the protein of interest. Junjie Shao is definitely correct that antibodies are not always great for this use and anti-flag maybe the better option.
I can only think of the possibility of an antibody binding just in the same region that would interact with the second protein. In that case there would be less co-precipitation.
The anti-tag antibodies are a historical idea. When no good commercial antibodies were available, you used a tag to detect your desired interaction. It is much more elegant to use specific antibodies and relevant protein concentrations e.g. isolate a specific cell type ex vivo instead of doing an artificial overexpression (sorry for the criticism on your Hela model).
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