I am trying to understand the Y2H screening method. I can understand how we can check if two specific proteins interact with each other. For example, if we want to check whether protein A and protein B interact, we fuse A with the Activation Domain (AD) of a transcription factor and B with the Binding Domain (BD) of another transcription factor. When the two fusion proteins are transfected into a yeast cell, the reporter gene is expressed only if the AD and the BD get together and form a complete transcription factor and that happens only if proteins A and B interact with each other.

However, I have seen claims that Y2H is a high throughput system, i.e., it can be used to detect several interactions at once. But most articles online that attempt to describe it, seem vague to me.

For example, suppose I fuse the "bait" protein to the BD and several different cDNA's to the AD and let the yeast grow. Next, I observe that the reporter gene is expressed. Doesn't this observation only imply that there exists some protein from the cDNA library that interacts with our bait protein? How do we know exactly which of the target proteins have interacted with the bait and resulted in the expression of the reporter gene?


1 Answer 1


You use a library of many yeast in which each expresses only one target, or 'prey' protein. Then you grow each yeast colony separately, for example in different wells.

It's definitely not a high-throughput method if you do it the old-fashioned way, i.e. making your own yeast library, or if you do it with only a few targets. But these days you can buy pre-made yeast libraries with thousands of target proteins, each separated into a well on a plate, then you run the experiment for all the targets simultaneously. With a visible marker you can then easily see which wells have produced successful interactions, and thus which targets interacted with your bait.


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