Two-hybrid; hybrid protein nuclear localization?
rocco at mail.utexas.edu
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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