promoter analysis

[mhollowa at epo.som.sunysb.edu]

In article <CJ7FL1.I85 at biobase.aau.dk> sjn at biobase.aau.dk (Soeren Jensby Nielsen) writes:
>1)   What is your preferred reporter gene - and assay?

I've been using the Seed and Sheen CAT assay.  Very inexpensive and, when 
done with the straight xylene extraction, much more sensitive than the 
normal Gorman TLC assay.  Much easier to quantitate.  Gene 67 (1988) 
271-277 (Also found in Current Protocols).  

I was recently informed of a very interesting secreted placental alkaline 
phosphatase reporter assay that is very easy, just cook the cell media 
(no cell extract necessary) for an hour at 65-70 degrees and do a very 
sensitive fluorescence assay or a less sensitive colorimetric assay.
Methods Enzymol 216: 362-8 (1992)

>2)   Have you got a favorite vector, and where can one get hold
>     of it?

The Promega CAT vectors are very good, easy to work with.

>3)   Do you use an internal transfection standard, or normalize
>     to total protein content.

You definitely need an cotransfected reporter to correct for efficiency 
if you are comparing nested deletions.  Nothing can substitute.

>I might add that this poor university is not equipped to do luci-
>ferase assays, so light is out of the question.

There is a method for doing luciferase in a scintillation counter.

Mike Holloway
mhollowa at epo.som.sunysb.edu