Luciferase reporter assay - (Aug/11/2008 )

Hello!
What is the principle of Luciferase reporter assay ? and how it can help to study :
Promoter structure
Gene regulation
Signaling pathway
Thanks

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Essentially you put the gene for the enzyme Beta-galactosidease into a genomic context

For promotor studies you would put B-gal downstream of your promotor of interest and the cells will produce B-gal as if it were the original gene under the same promotor. To this effect you can analyze how much expression occurs of your original gene based on how much B-gal gets created under the same conditions. you would then lyse the cells and add luciferin substrate and measure luciferase activity in a luciferometer. Otherwise you can add ONPG to the lysates which will turn yellow at a rate and intensity proportional to the expression of B-gal. You would measure yellow-ness in a spectrometer.

For gene expression it would be the exact same story. Replace the ORF w/ B-gal.

For signalling it's again the same story, except that you would have to have B-gal under the promotor that is targeted by the transcription factor your interested in. When the signaling occurs B-gal will be expressed, when it doesn't it wont.

All of these assays are highly quantifiable however, the caveat is that Bgal expression and activity isn't considered the most consistant biochemical tool. It is widely known that any assay that uses luciferase as a reporter currently has a more advanced, relable, precise, and acurate tool to use. If you describe youre experiment more specifically I can direct you to a more advanced technique.

Anything else?

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Hi Dr.MHB, I'm a postdoc that daily deals with luciferase with bioluminescence imaging. Of course, luciferase, as other reporters, can be very helpful to study lot of molecular mechanisms, not only confined to transcriptional regulation. Let me suggest you to follow the Luciferase Channell at my blog Reportergene

Don't hesitate to ask more detailed questions!

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