in-vitro coupled wheat germ system [Promega]

newsnet customer via methods%40net.bio.net (by customer At newsnet.com)
Fri Nov 24 19:08:46 EST 2006


>> 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.

I agree that the luminometer is by far superior than the naked eye. However, 
I don't plan to quantitate the amount of luminescence at this stage but 
rather if there is a positive or negative result (ie. light or no light in a 
dark room). But you may be still right in that the luminescene maybe too low 
to detect by eye. Anyway, I'm going to give it a go. Going to use a quartz 
quvette... that should help with the luminescene compared to a micro-tube.

Cheers,
ST 




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