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higherme
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I was reading a paper on yeast two hybrid, and I was wondering why a bait protein and GAL4 activation domain show growth on Leu- medium. I thought there should be no interaction since there is no prey protein?
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.
A YTH assay is a laboratory technique used to study protein-protein interactions. It involves fusing a protein of interest to a DNA-binding domain and another protein to an activation domain. If the two proteins interact, they will bring the DNA-binding and activation domains into close proximity, leading to the expression of a reporter gene.
Leu- medium is a selective medium that contains a limited amount of the essential amino acid leucine. In YTH assays, this medium is used to select for yeast cells that have successfully taken up both plasmids containing the fusion proteins. This ensures that only cells with the desired protein-protein interactions are able to grow, making the results more reliable.
The strength of a protein-protein interaction can be measured by the level of reporter gene expression in the YTH assay. If the two proteins strongly interact, there will be a higher level of reporter gene expression compared to weaker interactions. Additionally, the strength of the interaction can also be measured by the growth of yeast cells on selective media, with stronger interactions resulting in better growth.
No, YTH assays are limited to studying interactions between proteins that are able to enter the nucleus of yeast cells. This means that interactions between membrane-bound proteins or proteins that require specific cellular conditions for interaction cannot be studied using this method.
YTH assays have some advantages over other methods for studying protein-protein interactions, such as being able to study interactions in a more natural cellular environment. However, they also have limitations, such as only being able to study interactions between proteins that are able to enter the nucleus. Other methods, such as co-immunoprecipitation and surface plasmon resonance, may provide complementary information and can be used in conjunction with YTH assays for a more comprehensive understanding of protein-protein interactions.