Hi all,
I have a question regarding the expression level of the bait protein
vs the expression of target protein. As a bait vector I use the pAS-2
or a derivate in which expression of the GAL4 fusion protein is driven
by the native ADH1 promotor which results in rather high expression.
The AD-library was inserted into pAD-GAL4 (Stratagene) where expression
of the fusion protein is driven by a truncated form of the ADH1 promoter
which results in low expression (not detectable in western).
The Stratagene manual says about that:"Overexpression of the bait
protein
can result in a phenomenon called "squelching". When squelching occurs,
ecxess unbound bait protein (not bound to GAL4 UAS) binds target
proteins
thereby preventing the target proteins from interacting with the bait
proteins, which are bound the UAS. Consequently, transcription of
reporter genes is not activated and interacting proteins are not
detected."
What do you think about this? Shouldn't I combine those two plasmids
with
different expression level? Or can I disregard that?
Any suggestions will be appreciated.
Cheers
Ricky
******************************************************************
Frederik Boernke
Research Group of Molecular Plant Physiologie
Institute for Plant Genetics and Crop Plant Research (IPK)
Corrensstr. 3
06466 Gatersleben
Tel. 039482 -5 321
Fax. 039482 -5 515
e-mail: boernke at ipk-gatersleben.dehttp://www.ipk-gatersleben.de
