The discussion revolves around the yeast two-hybrid (Y2H) system, specifically focusing on the growth of yeast on Leu- medium when only a bait protein and GAL4 activation domain are present. Participants explore concepts such as auto-activation and the implications of using different carbon sources (glucose vs. galactose) in the screening process.
Participants express uncertainty regarding the mechanisms of auto-activation and the implications of using different media conditions. Multiple viewpoints on these topics remain without consensus.
Participants have not fully resolved the implications of auto-activation or the specific effects of using glucose versus galactose in the context of weak versus strong interactions.
Indeed and this can lead to false positives, it is always good to check auto-activation of the bait (or use stringent conditions that rule out auto-activation).higherme said:is it when the bait itsef can activate transcription without the prey?
In this case you would need to use a yeast strain that is Leu-auxotroph. You do the Y2H assay on a minimal media plate (laking Leu), so that the auxotroph can not grow. Only when there is an interaction between the bait and the prey does the promoter get activated, which should drive Leu expression giving rise to growth of the yeast.another question i want to ask is what is the differnce between doing the yeast screen on medium lacking Leu, either with glucose or with galactose? Is it because the prey protein is often induced with galactose? if there is a weak interaction, only growth shows on Leu- with galactose, whereas, in a strong interaction, both media with show growth?
thanks in advance.