Two-hybrid; hybrid protein nuclear localization?

Stephen Palmieri rocco at
Sat Apr 29 09:41:32 EST 1995

		Unfortunately, the Fields/Elledge system does not provide an assay 
system to determine nuclear translocation of your fusion protein.  On 
the other hand, the Brent system does provide a method to determine this 
that might be applicable.  It is more or less a competition assay system 
in which a plasmid containing, in your case, GAL4 is transformed along 
with your fusion protein construct.  What you are assaying for is a 
decrease in transcription of the lacZ gene due to competition of your 
fusion protein with GAL4 for binding to the GAL4 UAS.  It is not a 
dramatic change, only a 2 to 5 fold decrease.  I have not conducted this 
assay myself because I have a positive control for my particular fusion. 
Hope this helps.

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