Alpha Screen technology (from Perkin Elmer):http://www.perkinelmer.com/Catalog/Category/ID/AlphaScreen%20Assays%20and%20Reagents

Good luck!

1) GST-pull down (or Halotag or Co-IP) using recombinant proteins (expressed in bacteria or in rabbit reticulocytes), even S35-Met labeled if specific Ab for the target protein is not available.

2) Fluorescence resonance energy transfer (FRET), trickier than 1).

Use pierce label transfer technology if you have your protein in pure form. Then you can do strigent washes to remove indrectly interacting proteins and purify the direct interactors...

I would agree with the other posters who have suggested that you probably need to purify the proteins. If you do this, you can try other methods like surface plasmon resonance that will give you a lot more information as well. There are a lot of options once you have purified protein.

I'm guessing that you are probably working with eukaryotic proteins, and that purifying all of them will be either difficult or at least very time consuming. One approach to get round this would be to try non-dissociative mass spectrometry. This would require you to be able to get hold of a reasonable amount of your protein plus partners, freed from the pull-down beads. Non-dissociative mass spec. can help a lot by identifying the order in which parts of your complex fall apart - if you are left with your protein of interest with a single partner, it's a pretty good bet that they directly interact!

This is a pretty specialised technique, so you would need a good mass spectroscopist who knows what they are doing to help you.

If it is essential for you to study the protein in live cells, FRET studies done with GFP tagged proteins could work, however, this will require to tag both proteins with GFP tags.

PLA is good to try. You could quantify also with this technique.You need really good Primary Antibodies which are specific or as others suggested you could perform co-ip.

Maybe you could try the Yeast two hybrid system...? http://en.wikipedia.org/wiki/Two-hybrid_screening

1.Could you please specify the difference between a direct and an indirect interaction?

2. You may want to look into the TAP technology (http://en.wikipedia.org/wiki/Tandem_affinity_purification) which allows you to isolate protein complexes for subsequent analysis.

3. Direct protein-protein binding (as opposed to protein assembly into a stable complex) can best be studies by biophysical methods such as Surface-Plasmon-Resonance and Bio-Layer Interference but you need to have access to these expensive instruments and you need to have your target proteins purified.

4. Native PAGE or BlueNative PAGE can also be of use to demonstrate a shift in mobility upon the presence of an interaction partner (but you'd need a control without the interaction partner for comparison). Analytical Size Exclusion Chromatography (Gelfiltration) can also be useful.

5. A lot will depend on your target protein and your possibilities to manipulate it

ELISA-Style binding assays using recombinant proteins. With this method you could also measure the affinity of the proteins to each other, but actually its super-in vitro....

I would recommend MicroScale Thermophoresis (MST). It is a very new biophysical methods which allows you to study your protein - protein interactions directly in free solution. With MST you do not have to immobilize your proteins like you have to do with SPR or BLI (immobilization can introduce strange artifacts).

I have to say that I am biased as I am one of the inventors of MST. What we see is that the leading scientists are all moving to MST as our approach is much more simple and robust. MST needs much less material than all the other methods and it is very easy to learn: after 30 minutes you will know how to use our Monolith devices. MST also allows you - in the case of fusion proteins- to measure your unpurified samples directly in your lysate.

http://www.jove.com/video/50541/protein-purification-free-method-binding-affinity-determination

Dear Philip, please also mention that you need to label your protein for MTS or you need to produce it as a >>fluorescent protein fusion

Yeast two hybrid system, Pull-down, SPR or FRET, which one of them depends on your resources, equipment and expertise. With any of them you will need to clone your proteins and in some cases previously purify them. Good luck!

If you can make 100 ug amounts of your partner proteins then I suggest that you consider Small angle x-ray scattering or SAXS. SAXS depends upon the square of the number of electrons in a complex so it is very sensitive to detecting the formation of protein-protein interactions. SAXS can monitor interactions in solution under near-physiological conditions with tiny volumes and 1-2ug/ul concentrations at the level of 96-well plates (Nature Methods 6:606-12, 2009). One can directly determine mass from the scattering experiment without knowing the protein concentration and without requiring a fully ordered protein , so this method works on flexible complexes or even on intrinsically unstructured proteins (Nature 496, 477-81, 2013). You do not need your own equipment as you can send samples by Mail-In as part of NIH-funded efforts (see http://bl1231.als.lbl.gov and S. Classen et al., J Appl Crystallogr. 46,1-13.)

Dear Markus, I partially agree with you: to identify a protein that interacts with my target protein I would also use Mass spectrometry. However, to prove the direct interaction, I wouldemploy MST using the purified proteins. It only requires small sample volumes and concentrations, you can measure free in solution, it's straight forward in handling and in addition you will obtain not only the information IF the proteins are binding, but you will also be able to determine the affinity!! I guess that's quite an advantage :-)

SPR, photo-luminescence, electrochemical and some other techniques are useful. An example based on combination of few of them in one system is attached.

Article Comparative study of Surface Plasmon Resonance, electrochemi...

You can very easily and elegantly determine the unknown protein interactions with the Next Generation Sequencing Yeast Two Hybrid method in vivo. The NGS Y2H works best for binary interactions. It excludes the false positives that you will get with conventional Y2H screens, so you wont have to spend your time and money later verifying hits. the problem with MS is that although it gives you the hits for binders, those may not be functional. NGS-Y2H will reveal the functional interactions. It also reveals the low abundance proteins from weak enrichments.

Its also possible to map the binding with Next Gen Seq Y2H, if you would like to know where exactly the binding happens (domain mapping).

You can look into the technology and services of Next Interactions in Richmond, CA.