Luciferase assay and luminometer - Technical details (May/06/2008 )

Hi,
I'm about to start a promoter analysis using pGL3 luc and pBgal as internal control.
I have a Firefly luc kit from Biotium and I'm planning to do the experiments in HeLa cells.

As I've never done this before, and don't know anyone who did...I have some basic doubts about preparing and doing the reading. So if any of you has been there before I hope that could help me.

First of all, I'm using a plate luminometer, so I'd like to know if plates are standard or have to be special for this assay.......I read something about opaque plates.......is this the kind to use? and the size of the wells is just according to my needs (how many different conditions I'm studying).......this luminometers read a 24 wells plate as well as a 96 wells plate?

Then, according to the protocol, I transfect cells with the pGL3luc -my promoter, and with the one used as internal control (in my case pSV-gal).
It's not possible to do the readings in the original plaques of the cells, right?

After cell lysis (using passive buffer) I transfer to a tube, and samples can be stored at
-70....and then I take just an alicuot of each and transfer to the plates to measure luc activity, is that OK?
The protocol says to use 500ul of lysis buffer per well in a 6 well culture plate........so this is the stock of cells in each condition...... then the volumes to use in the luminometer are 20ul of cells lysate and 100ul of assay solution.....my question.......... are this volumes independent of the size of wells using?? I mean is it the same if I use 20ul in a 96 wells plates, or in a 24 wells plate??

One last thing, do I have to do a standard curve previously? is it necessary? how trouble and time demanding is it??

Ups.. that wasn't the last: do you know if there is any guide or paper about all this to have things more clear!!

hope you can help,


bio..

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I'm not an expert on this but I have done a few Lucys last year. So i will try to answer what i can..

About the plates - we had special 96plates for use with the lumiometer. they were non-transparent in black or white. I don't know if it is of importance but I could image that if you try to measure and have strong signal(light emission) in the first well but in the next a weak a transparent plate could lead to problems. but best have a look at the manual of the photometer or call the support service. probably you will also have to program the luminometer according to your experiment...however this was made by other people from the lab so i can not say much about it.

You mean in the untransfected cells? No - there won't be any signal.



Freezing worked fine at this step.



I think you should take less lysisbuffer if you have smaller wells. I can't really say much about it as the protocol was already established in the lab when i was there, besides we used dual glow from promega. anyway the amount of lysisbuffer shouldn't play a role you can make some preexperiments and just try to find with what amount you get a good signal and then keep it constant with always using assay solution as indicated.


unfortunatly i can not give you more information but maybe that helped a little bit.

One more thing - check your protocol for light sensitive reagients. Some steps then should be carried out in the dark...

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thank you very much ooolex.!!

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biotech
I dont think you need a standard curve to express your results. As far as I know, you need to normalize your signal with transfected internal control, then express as fold induction with control.

Hhh Im also agreed with ooolex that "the optimization of volumes and other experimental set up" is depend on cell type, cell density, inducers, luciferase kits and many other steps, so it should be determined empirically.

Cheers
Thapa

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