I was unaware of a *variability* in luciferase half-life such as you=20
mention it. What magnitude of variability are you talking about? from=20
hours to days? What do you mean by "changes with transfection=20
conditions"? Do you mean, for example, between cells transfected with CaPO4=
versus cells transfected with liposomes? Or, which would be a lot more=20
troublesome, between cells all transfected the same and subjected to=20
different culture conditions (e.g. hormonal, etc.) as part of an=20
experiment? Is this variability documented somewhere?
The SEAP model seems attractive, although the idea of concluding to a 100%
repression when one observes no change at all, while correct as you=20
present it, is a bit of a mind twister.
Salut la France!
On Tue, 8 Oct 1996, Florence CABON wrote:
> Date: Tue, 08 Oct 1996 15:44:08 +0200
> From: Florence CABON <cabon at infobiogen.fr>
> To: methods at net.bio.net> Subject: Re: Luciferase half-life?
> I am looking at transcriptional repression of a promoter. I had the same
> problem. I tried several times to measure luciferase half life but it
> changes with the transfection conditions and maybe with the weather
> outside :-). I resolved this by... changing luciferase to SEAP
> (secreted alcaline phosphatase). Tropix, Clontech and ICN sell kits to
> detect SEAP by luminometry. The test is very sensitive and even more
> than luciferase so you can work with very little cells. The advantage is
> that SEAP is very very stable, even at 37=B0C. So when you want to know
> the kinetic of a reaction it is very simple, you take aliquots of the
> medium (it is secreted) You keep your aloquots at 4=B0C and when the
> experiment is finished, you measure the seap activity. You can then
> calculate the differences between to time points. If there is no
> differences, you've got a 100% repression of the promoter at that time.
> The problem is that you have to reclone your favourite promoter in front
> of SEAP.
> Florence Cabon.CNRS Villejuif, France.cabon at infobiogen.fr> =3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=
Dr. Louis H. Ferland
Centre de Recherche, Hotel-Dieu de Montreal
Dept de Nutrition, Universite de Montreal
Phone: (514) 843-2757 FAX: (514) 843-2719