in-vitro coupled wheat germ system [Promega]

[DanSolo]

> Hi,
>
> I want to visualise sea pansy (renilla) luciferase enzyme/protein produced from the in-vitro coupled wheat germ system from Promega. Here are my options:
>
> Option 1. AUTORADIOGRAPHY of radiolabelled luciferase [s35 Met] - lab cannot work with radioactive material, so this is ruled out.
>
> Option 2. LUMINOMETER to detect luciferase luminescence  - luminometer is not functional at the moment, so this is ruled out.
>
> Option 3. WESTERN BLOT using antibody against luciferase - this is a viable option.
>
> Option 4. Visualisation of luciferase luminescence by eye/microscope in a dark room - this is a viable option and quickest. Not sure how much protein (pg,ng,ug,mg??) you need before you can see some light glowing in the dark??
>
I don't think you'll ever see the amount of luminesence that a
luminometer detects by eye, especially with the renilla luciferase
which we find is nowhere near as strong as the firefly. S35 would label
loads of other proteins too, unless I misunderstand your protocol. I'd
go for the western blot, and if you can find a monospecific antibody
you also could try dotblotting it.