I am trying to understand the Y2H screening method. I canunderstand how we can check if two specific proteins interact witheach other. For example, if we want to check whether protein A andprotein B interact, we fuse A with the Activation Domain (AD) of atranscription factor and B with the Binding Domain (BD) of anothertranscription factor. When the two fusion proteins are transfectedinto a yeast cell, the reporter gene is expressed only if the ADand the BD get together and form a complete transcription factorand that happens only if proteins A and B interact with eachother.
However, I have seen claims that Y2H is a high throughputsystem, i.e., it can be used to detect several interactions atonce. But most articles online that attempt to describe it, seemvague to me.
For example, suppose I fuse the "bait" protein to the BD andseveral different cDNA's to the AD and let the yeast grow. Next, Iobserve that the reporter gene is expressed. Doesn't thisobservation only imply that there exists some protein from the cDNAlibrary that interacts with our bait protein? How do we knowexactly which of the target proteins have interacted with the baitand resulted in the expression of the reporter gene?
I am trying to understand the Y2H screening method. I canunderstand how we can check if two specific proteins interact witheach other. For example, if we want to check whether protein A andprotein B interact, we fuse A with the Activation Domain (AD) of atranscription factor and B with the Binding Domain (BD) of anothertranscription factor. When the two fusion proteins are transfectedinto a yeast cell, the reporter gene is expressed only if the ADand the BD get together and form a complete transcription factorand that happens only if proteins A and B interact with eachother.
However, I have seen claims that Y2H is a high throughputsystem, i.e., it can be used to detect several interactions atonce. But most articles online that attempt to describe it, seemvague to me.
For example, suppose I fuse the "bait" protein to the BD andseveral different cDNA's to the AD and let the yeast grow. Next, Iobserve that the reporter gene is expressed. Doesn't thisobservation only imply that there exists some protein from the cDNAlibrary that interacts with our bait protein? How do we knowexactly which of the target proteins have interacted with the baitand resulted in the expression of the reporter gene?
