A Q: for transfection gurus. We are measuring promoter activity using
luciferase gene
as a main reporter, fused to the putative promoter fragments, and
CMV/beta-galactosidase
gene as a second, normalizer activity. Both are introduced by lipofection
into an aortic cell
line. Luciferase activity is measured using a Promega kit, and the b-gal
is measured via
a chemiluminescence based kit from Tropix (GalactoLight). Before
measuring the latter
activity we heat-inactivate the endogenous activity. What we get is that
for a given construct
the luciferase activity in a 3X transfection series is relatively constant
( within a factor of 1.5), but
the respective set of b-gal activities is variable within a factor of 3,
which ruins the experiment.
Any ideas? Has anyone used GalactoLight with success?
--
Alexander Kraev, PhD
Biochemie III, ETHZ Zurich
Phone 41-1-632-31-47
Fax 41-1-632-12-13
e-mail kraev at bc.biol.ethz.ch
