Ferland Louis H. wrote:
>> Martin, John, and all the rest of you, Netters,
>> The half-life of the protein is only half of the story. I am in a
> position similar to John's i.e. I want to verify possible (expected)
> rapid inhibition of a transfected promoter driving the LUC reporter. But,
> to have an idea of whether LUC is useable for this type of experiment, we
> need not only the half-life of the LUC protein, but also of its mRNA.
> Does anyone have any data on this?
>> We're just going to give it a try...
>> - Louis
>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°C. 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°C 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
Florence Cabon.CNRS Villejuif, France.cabon at infobiogen.fr