Troubleshooting & EGY188 needed in Camb

Erica Golemis EA_Golemis at fccc.edu
Mon Dec 4 16:00:41 EST 1995


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




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