y2H problem - (Feb/27/2006 )

Hi all,

I use the clonetech yeast two hybrid system for detecting interactions. I sreen my bait against a single protein (prey). All the reportergenes are expressed suggesting that my bait and prey interact. Their are no signes of autoactivation. However, when my bait is cotransformed with a unrelated prey all the reportergenes are also expressed, but in a less strong manner. How can I get rid of this (false) positives?
kind regards

You say that your bait does not autoactivate, I am assuming your tested this by transforming with you bait protein alone. So your problem could be:-

1. Your negative control ("non-specific" prey) binds DNA directly and non-specifically and so turns on the reporters
2. Your bait protein might be "sticky" - i.e it binds all/many protiens

Addition controls would be the empty bait (GAL4 BD only) + your negative AD-control, and your bait + empty prey (GAL4 AD only).

Which unrelated prey are you using?
Are you sure you can use as negative control?
I was using lamin C as bait to control for fortuitous interaction between an unrelated protein and the prey.
lamin C didn´t interact with the protein I used as bait but it interact with another protein I used as positive control (one known interctor I have used as positive control prey). I found out that indeed there was already a paper describing the interaction of Lamin C and that protein.

Do a search on internet to know more about the protein you are using as negative control?

I 've used the large sv40 antigen as negative control. I don't think its related to my protein.
Is there anything I can do if my protein is 'sticky'.

Why have you chosen the SV40 has negative control, where did you read that?
Was in an article?You said that the bait and unrelated prey also activate expression of reporter genes. You should trnasform the yeast only with the unrelated prey to check if it has transcription activity/autoactivation test. Only if it doesn´t autoactive you can use has negative control but even then you should be careful. You assume your bait doesn't bind to the SV40 but maybe it does.