Y2H interaction is nuclear and therefore i doubt it will work.

My suggestion would be to synthesize a biotinylated peptide and do a pull down with MS peptide fingerprinting of the pull down bands.

Thank you Prof. Huyton. The candidate proteins were obtained by  MS peptide pull down assay and we would like to confirm the interaction by a more solid method.We are looking forward to your suggestion.

Perhaps the Y2H would work if you are using just the 20aa but it is also reliant on having the prey well expressed in the library and many many other factors.

It is probably much easier to make:

Native  immunoprecipitations (with or without crosslinking, depending on the strength of the interaction) to confirm the binding.

co-localization (immunofluorescence)

Mutagenesis of thepeptide to show the interaction is specific.

Your suggestion raised another question whether co-immunoprecipitations could detect the interaction between 20aa C-terminus peptide and a much bigger protein (about 400aa). Thank you very much.

I agree with Dr. Huyton that there are multiple problems with the Y2H methodology.  Besides the library considerations and other factors, I have found frustration with the number of false positives generated.   My experience suggests to me that it is not worth the time, energy and expense spent on false positives.

I also agree that the Y2H might give information on the interaction of the peptide, but I find his alternate suggestions simpler, quicker and more reliable. Besides these techniques, there are other possibilities for you to try that are highly accurate and can be multiplexed.

Of course, I am biased towards bead based assays.  When used in flow cytometric methods using microspheres, such assays can be used to assess peptides that interact with intracellular binding partners such as ligands, G proteins and kinases.  These methods have been established and include multiplexing and sorting as additional possibilities for the beads.

Flow cytometry has become a key tool in the area of GPCR research.  In your investigation, it would add the capacity for discrimination of multiple particle populations and high throughput.

I do not know the resources available to you, but I would add that the utilization of flow cytometry would reduce time, cost  and vastly decrease the amount of sample material needed from your source materials.  You can alternate between cellular and molecular analysis or perform both together.

It is a little old, but I might recommend the following as an overview: Techniques: GPCR assembly, pharmacology and screening by flow cytometry.  I believe Dr. Bruce S. Edwards has posted the article here in Research Gate.

My best wishes on your investigation of the c-terminus peptide.

Thank you Prof. Cimino, your suggestion is very valuable. The flow cytometry method is good for screen GPCR interaction protein and molecule, but in our investigation  the candidate proteins had been screened from tissues by MS pulldown assay and we would like to validate the results. We thus wonder which kind of method might work, mammalian two-hybrid or coIP.