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Normalising luciferase activity


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#1 Nay

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Posted 20 February 2005 - 05:55 PM

We have recently stably transfected some PC12 cells with our promoter luciferase construct, and we are about to do some luciferase assays using various stimulants.

Because of the time needed to stimulate the cells, we were unable to go with transient expression of our promoter construct... hence the stable lines. However, now I am not sure how to go about normalising the luciferase activity. When performing transient transfections, I would usually co-transfect the cells with a beta-gal plasmid and perform a simple beta-gal assay for transfection efficiency, but I can't do this in my stable cell lines.

Is meauring total protein by Lowry or Bradford assay a suitable way to normalise? :)

#2 badcell

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Posted 21 February 2005 - 02:40 AM

I've never done stable transfections for gene reporter assays, but I would say that protein concentration would be a good way to normalise your samples. In theory, you would have a basal level of luciferase activity in the transfected cell line which (when the cells are maintained under the same conditions) will only depend on the number of cells and thus on protein concentration of your lysate. So the luciferase/protein ratio of your cells should be a characteristic value. This constant would be altered when you stimulate the cells, indicating that the luciferase activity per cell has increased/decreased.
Hope that helps!
Science is a wonderful thing if one does not have to earn one's living at it
(A.Einstein)




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