All the methods has advantages and disadvantages. As you don't know the potentials prey you could not use FRET. For Co-IP and Protein Affinity you should latter use mass spectrometry for identification of proteins. Using these methods you detect complex of proteins, but is difficult to assure which one is the protein from the complex that bind to your protein of interest. I suggest you to use Y2H to identify your possible prey proteins and then confirm by Co-IP followed by WB. For Y2H there also some disadvantages that you could find in published reviews.

The pull-down method is frequently used to identify binding partners for immobilized ligands. It's basically the same as co-ip, except without necessarily using antibodies. One protein is immobilized on a resin, the cell extract is mixed with the resin, and, after washing, the bound proteins are eluted, subjected to SDS-PAGE, the bands are cut out, and mass spec is used to identify the proteins after proteolytic digestion.

Hola Julio,

Thanks for your answer and the suggestion which were informative. What if we use HaloTag technology of Promega? Which can be fused to the bait protein and binds covalently to a HaloLink resin. Then, a forceful wash and elution may reduce the complexity of the web of proteins to our only desired interacting proteins. Does still the problem with detecting our potential prey proteins by spectrometery exist? It's just a guess and I'm not sure about it! It also has no mammalian homoluge so less background interference in WB is detectable.

Hello Adam,

Thanks for the answer. I'll definitely consider pull-down method since I read a paper about it and it was easy to use. It can be considered as protein-protein equivalent of the Y2H. Does the immobilization of the protein must be done with GST fused to the bait protein which can bind to the glutathion particles? or it vary in different methods how to immobilize the protein?

SDS-PAGE, In-gel or in solution trypsin digestion, LC-MS/MS, protein identification

The immobilization of the bait protein can be accomplished in a number of ways, including affinity tags such as GST, poly-His, Avitag etc; chemical biotinylation; or direct crosslinking to the resin.

An advantage of using direct crosslinking to the resin (e.g. CNBr-activated Sepharose) is that the target proteins can be eluted from the resin with SDS-PAGE sample buffer without also eluting the bait protein. The disadvantage is that crosslinking may interfere with the protein-protein interaction.

Dear Adam,

Many thanks to your precious time and the valuable information that you kindly rendered which were really insightful for me to plan my research more carefully. Frankly, I learned too much. Thanks.

Hello Silvia,

Thanks to your answer and your time.

Very Welcome Saeid, thank you for your question and followers answers.

I am curious to know how we can investigate the PPI in redox signaling partners regarding this fact that their interaction is fleeting and unstable! In that case, the PPI is fast and is performing only via transfer of electron! but the two partners interact.

please any one these protocols send it to me  

Co-immunoprecipitation (Co-IP)

Protein Affinity Purification Methods

Two Hybrid Assays (Y2H)

Forster Resonance Energy Transfer (FRET)

Far Western blotting

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