Hi,
I want to check interaction strength between my protein of interest and several candidate interactors using Y2H (TakaraBio, previously clontech). If I follow manufacturer's instructions which is meant for library screening, I will need to transform separately bait into Y2HGold strain and prey into Y187.
However, in relevant publications, authors co-transformed two constructs into one yeast strain (AH109).
Selection pressures are the same between the two methods (W, L, A, H).
1). Can I test protein interactions using mated/diploid yeast rather than yeast co-transformed with 2 plasmids?
2). Are there reasons why one method is preferred over the other? Thanks.
co transformation is more simple but you will have less co-transformant so you need rather clean DNA; it depends also how many prey you have to screen it can be convenient to make a strain with your bait gene and then transform it with your preys. I did 2HY long time ago and never use mating (of yeast)
I used to do sequential transformations rather than matings. I would put in each bait then later transform in each prey (as suggested above). I had a small number of baits and preys so it was easy to make all the desired combos.
I always transform both bait and prey in AH109 and plate in -LT minimal media. Most of the time I will get transformants and can proceed them for higher stringency selection. However, some interactions may be toxic so in case co transformations do not work, matings can be done
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