Co-IP of wnt pathway - (Nov/22/2013 )

Hi,

I have never doen Co-IP and would have to perform  one soon. I would appreciate all advice in this regard.

I want to check if my drug of interest affects the GSK3B/Axin/b-catenin complex. But I’m not sure where to start. Do I pull down with GSK3b and check if Axin and b-catenin are holding on?  Also, can all mono clonal antibdodies be used for Co-IP? I have GSK3B and Axin from Cell Signalling and the manual says they can be used for IP?

Many thanks

SF

If the antibodies have been used for IP, it is likely that they will work.  However, in general, polyclonal antibodies are better for IP as they have more than one binding site on the protein of interest, but this doesn't mean that monoclonals can't be used.

I don't know anything about the wnt pathway, so I can't really talk about specifics... however, in general if you think something is affecting a complex formation then co-IP should work as you suggested.  There are other ways of looking at this too, but less directly - such as looking at activity (reporter assay?) of downstream targets.