DNA binding by GAL4-fusion proteins

Kannan Thiru kannan_thiru at hotmail.com
Fri Mar 20 11:53:03 EST 1998


Hello!

Does someone know the sequence of the GAL4 DNA-binding element (UAS-G)
that could be used for gel-shift analysis.

I am doing structure/function analysis of a tissue-specific
transcription factor by deletion analysis using GAL4-DNA binding domain
fusions.  In order to show that the GAL4-fusion proteins are actually
made in transfected cells, I am planning to do a gel-shift assay, using
nuclear extracts from transfected cells and the GAL4 DNA-binding element
(annealed and labeled synthetic oligos) as probe.

Would someone be able to tell me the sequence I could use, and the
optimum binding conditions.   I would appreciate an early response.

P.S.  I have difficulty in seeing the proteins on a western blot
(probably due to low level of expression).   Since the gel-shift
analysis is more sensitive, I would like to try it.  Further, the
gel-shift assay will not only indicate whether the proteins are made,
but also show whether they can bind to the DNA.

Thanks.

Kannan Thiru, PhD
630E, Dept of Molecular and Human Genetics
Baylor College of Medicine
Houston, TX 77030.

Phone: (713) 798-5488 or 6873
Fax:   (713) 798-1465



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