Jens Riese <jr2 at mrc-lmb.cam.ac.uk> wrote:
>> I am performing a two-hybrid assay using the Brent lab methods.
> Unfortunately, my bait activates the reporters sometimes. Does anyone in
> Cambridge have a leucine reporter that is less sentitive, such as EGY188
> or EGY194? My bait also seems a bit random when many colonies are
> tested, with respect to both the leucine and the lacZ selection. Is this
> normal? and if so could I just pick a colony that gives low background?
> Any help or advice would be greatly appreciated. Thank you very much in
> advance.
> Xiang Yu (LMB, Cambridge)
>>Dear Jens,
The strain you want to use is EGY191 (a better-behaved relative of
EGY188). I will be happy to send it to you, if you supply your address.
Concerning the heterogenous activation of reporter phenotype: the
greatest likelihood is that the protein itself is being heterogenously
expressed. In that case, the less-activating transformants are probably
making less protein, and it is not necessarily a great strategy to
use them because following library transformation they can develop
background. There are various options you can follow, depending on
your preferences. In any case, I would recommend doing a Western to
compare you activating versus non-activating constructs before making
a decision.
regards-
Erica
