I am currently assaying activity of different regions of my favorite
protein fused carboxyterminal to the GAL4 DNA binding domain. In the
cases where the resulting construct is unable to activate transcription
of the GAL1-lacZ reporter that I am using, Is there any reason to doubt
that these proteins are capable of binding the GAL1 UAS? They still
contain the intact GAL4 DNA binding domain and I have verified protein
production by Western Blot analysis.
In the event that I have to test for DNA binding, is a gel retardation
my only option? Are there any constructs that contain overlapping
promoters such that binding of the GAL4 DNA binding domain will cause
loss of activity of a reporter? If so, can it be sent to me? Any
information regarding the questions above will be greatly appreciated.
Thanks in advance.
Shelley Ann des Etages
Dept. of Micr. and Molec. Gen.
185 S. Orange Ave.
Newark, NJ 07103.