Help: beta gal reporter activity unreliable

A.V. Peeters avp at sun_land.sun.ac.za
Thu Oct 31 00:35:04 EST 1996


Alexander Kraev wrote:
> 
> 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

We've had exactly the same problems with the same reporter plasmids
(luciferase and beta-Gal) in CHO cells : nicely grouped luciferase
results but useless beta-Gal activities. We tried using cmv beta-Gal as
well as pSV beta-Gal, but found no significant difference (we don't use
a kit for the beta-Gal assay and we do NOT heat-inactivate). We also
tested the constructs at different ratios for transfection - without
success. We've been struggling for some time now and hope to get
consistent results with the dual-luciferase reporter assay from Promega.

Does anybody have any idea what might be the cause of the beta-Gal
fluctuations ?



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