Does somebody has experience of Luciferase assay?. Actually my gene of interest is present in a vector also harboring GFP sequence. So transfection of this vector also results in expression of GFP protein along with my gene. Can I do luciferase assay with overexpression of such vector?. Green fluorescent protein (GFP) can induce any unwanted effect on luciferase activity?.
Thanks
If you make sure that you use an appropriate control plasmid (expressing GFP but not your gene of interest), it should be fine to use that plasmid for overexpression. We have done it and it worked quite stable. Alternatively, you can just sub-clone your gene of interest to another vector lacking a fluorescent marker.
Best,
Cindy
Hi Sajjad,
in our group we routinely perform luciferase assays using the Dual Luciferase assay kit from Promega. The vast majority of our genes of interest are cloned into vectors harboring a GFP or YFP tag which we then overexpress in human cell lines. We have not had any problems with our luciferase assays using these constructs, and have tested the effects of the tag by repeating the assays with our genes in un-tagged vectors. In our hands, there were no differences in luciferase data using either GFP-tagged or un-tagged vectors to overexpress our protein of interest.
I would still test the effect of the GFP tag on localization/expression of your protein just to be on the safe side because of the effects of this tag on protein stability (i.e. we sometimes see protein aggregates instead of diffuse staining depending on whether the tag is C or N-terminal to our gene of interest).
Hope this helps,
Pela
Hi,
Thank you very much Cindy Körner and Pelagia Derizioti. I strongly hope your answers will be useful for my experiment.
@ Pelagia Derizioti, could you expalin little more about protein stability please. How can I check the stability of my protein tagged with GFP?
Best Regards.
Hi Sajjad,
you can check the effect of the GFP tag on your protein simply by looking under the fluorescence microscope for example- does the addition of the tag lead to aggregates in the cells that are overexpressing the protein? Again you would have to compare with untagged versions, or even endogenous protein (if your cells express your protein of interest endogenously)- in this case you would have to stain for your protein using an antibody. In my experience, we very rarely see an effect, but have had problems in some occasions when we tried cloning our protein in a vector in which the GFP was C-terminal to our protein- this led to big aggregates in the cell, compared to normal diffuse expression patterns when the tag was N-terminal to our protein.
Hope this helps!
Pela
Dear Pelagia,
Thank you very much. Yes, I understand. Definitely your experience will help me.
Best Regards
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