genecutl at mendel.berkeley.edu
Wed Jun 22 11:40:31 EST 1994
In article <Charolyn_Babichuk-220694132925 at mac06.biochem.ualberta.ca>,
Charolyn_Babichuk at darwin.biochem.ualberta.ca (Charolyn Babichuk) wrote:
> I'm using firefly luciferase as a reporter gene in mammalian T lymphocytes.
> Although the B-gal standards work fine, the luciferase activity seems to
> be so variable from experiment to experiment. I know that the enzyme is
> sensitive to light and agitation. I have been using PMSF and DTT in the
> triton lysis buffer but if anyone knows of anything or any harvesting
> manipulation that can seriously affect the enzymes stability, please e-mail
> me and maybe we can discuss this problem.
> Blue Skies!
> Charolyn_Babichuk at darwin.biochem.ualberta.ca
I've had similar problems. I tried using luciferase as an internal
control for my CAT assays in Schneider cell transfections. I use
a freeze-thaw protocol to lyse the cells and split the extract into
an aliquot for CATs and one for luciferase. It turned out I got higher
variability in my luciferase assays than in my CAT assays.
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