Thanks for your piece of advice. I am not familiar with the methods you suggest, do you have any source I can use to get more informations?

Hi, may be you can try Bimolecular fluorescence complementation (BiFC) but this method is much more suitable to confirm a putative interaction.

BiFC is perfect to validate putative interactions, but it comes in a second phase. What I need first, is a screening methods, to identify interactions I don't know anything about yet

Yes, I guessed and it is true! Perhaps pull-down assay can be an alternative of Y2H although for me Y2H is Ok as first choice.

For screening your transmembrane protein against cytoplasmic proteins, I would truncate the transmembrane domain and perform Y2H library screens with a standard prey library eg from Clontech, then the hits you see from this screen can be confirmed in alternative methods such as split YFP, luciferase etc. We have done this a lot as we work with transmembrane proteins in the lab and interactions with truncated proteins seems to be quite accurate. 

Hi Stefano, is this a fishing expedition or do you have specific candidate proteins in mind that you suspect are interacting  with your transmembrane protein?

If you are looking for specific proteins and you have a good antibody against your transmembrane protein, you could try co-immunoprecipitation. There are plenty of references for that:

http://scholar.google.com/scholar?as_vis=1&q=%22transmembrane+protein%22+%22co-immunoprecipitation%22&hl=en&as_sdt=1,21

It will be a lot more difficult if you are looking for novel ligands that bind to your receptor.

You can identify the trans-membrane and cytoplasmic interacting proteins of a trans-membrane protein by Split-Ubiquitin based Y2H system itself but prey libraries to be of good quality. Further validation by the above suggested downstream methods like Co-IP and BiFC and be done. I have performed Y2H screen with Split-ubiquitin based method and it works great. Now days good quality prey-libraries are also available fro various companies that makes work very easy.

Good Luck

Thanks to everybody for your ideas. First of all, I work on plants. Second, it is a fishing expedition. I'm looking for plant pathogen interactions and, to my knowledge this would be the first study of this kind for the pathogen I am talking about. 

Thanks particularly to Kamal Kumar, I didn't know that split-ubiquitin 2 hybrid can be used also for interactions with cytoplasmic proteins. Good to know, it might be the solution...if I manage to obtain a good library!

All the advices about validation of specific interactions (BiFC or immunoprecipitation) are usefull for a second phase of the work, so thanks everybody!

Looking at your profile it seems that you are trying to identify interacting partners of plant defense "silver bullet" protein MLO. Earlier Dual-systems used to market the split-ubiquitin kits but now it has shifted its product line so you can contact Dr. Igor Stagljar, University of Toronto. Good luck..

Hello Stefano,

For the Y2H with the transmembrane protein, Split-ubiquitin system is well suited! However, for the cytosolic protein, it might not be. Here are a reference I found were they clearly say that Split-Ub is not suited for soluble protein (http://www.ncbi.nlm.nih.gov/pubmed/18428659). However in this paper (http://www.ncbi.nlm.nih.gov/pubmed/12112840), they explain that you can use the Split-Ub system if you first modify the bait (fatty acid modification).

I did a screening for a plant soluble protein during my PhD using the Gal4 system (pGBKT7 and pGADT7 vectors, available from Clontech). cDNA librairies from plant tissues are available for that kind of screening.

Good luck for your work!

As my paper was mentioned I feel inclined to comment. 

1. As far as I know Dualsystems has sold the SUS patents to Hybrigenics in France

2. Even though the term "Membrane yeast two hybrid" is widely used for the Split-Ubiquitin System (propably as a sale's pitch) I agree with the authors of this great review (http://www.ncbi.nlm.nih.gov/pubmed/22688816) that it is NOT a two-hybrid approach but split-complementation. (OK, that was nit-picking but I had to do it)

3. You can use soluble baits - at least sometimes. You would need to use the OST-based system that was also published by the Dualsystems people (http://www.ncbi.nlm.nih.gov/pubmed/17612295). I have also incorporated that into 'my' vector system hence with met-repressible promoter rather than a constitutive one and am about to publish this. But it is a hit-and-miss system. With one bait it works with another one not. Naturally, the companies will tell you differently but I can show you examples...

You can contact me if you need more details.

http://www.ncbi.nlm.nih.gov/pubmed/22688816

http://www.ncbi.nlm.nih.gov/pubmed/17612295

No system is full proof that is why we perform interactions with y2h, pull-down , BiFC, etc.  I like the DualSystems kits  (although sold now to Hybrigenics that may use it for Y2H services). DualHunter kit i regularly use & it works superb.