I have been doing research on interactions between three membrane proteins in skeletal muscle. .I want to experimentally determine whether the interactions between these proteins are mutually exclusive or can occur simultaneously. Basically, protein a interacts with b, protein a interacts with c, and protein b interacts with c. I want to determine if these are individual heterodimeric interactions, or if a three-way complex can form. Most of what I have done is using whole muscle homogenates or transfected cells. Any ideas are would be helpful. Thanks!

 

PFD

There are probably more elegant solutions, but the first that comes to mind:

 

Crosslink and do pull downs for each of your three proteins. Then probe with an antibodies for the other two proteins. So say you do a pull-down with A. Probe then with an antibody for B and then with an antibody for C. If you see a signal for B, but not C or if you see signals for B and C at different size ranges, then that would suggest a mutually exclusive interaction. If you see a signal for both at the same size range, then it suggests a complex.