s.d.huen at bham.ac.uk
Thu Jun 23 04:45:36 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.
Strange. We use Jurkat and don't observe that problem usually. Luciferase
variation in my system seems to be more from cell abuse and consequent
changes in promoter activity than luc itself.
Anyway, here's my easy-to-use, no freeze-thaw protocol for what it's worth.
Wash cells. Lyse in 500 ul of lysis buffer (100mM HEPES, 4 mM MgCl2, 5 mM
DTT, 2%Tx100). To assay luc, add 300 ul to 30 ul 25mM ATP in scint. tube.
Put into machine to squirt 100 ul 0.5 mM luc in glycylglycine buffer. Heat
remainder 50 degC for an hour. Assay bGal by GalactoLight (we make it up
Very incidentally, if you do you tissue culture experiments at 33 degC, you
will get 10X more luc signal than at 37 degC. Remember you heard this here
first! Prolly luc stability in mammalian cells is temp-sensitive.
David "Lazy Bazza" Huen, Temporarily UnEmployed Biologist.
More information about the Methods