Hi,
I have performed Luciferase assay more than 5 time separately. I transfected the 293T cells as well as HeLa cells with empty vector and vector harboring my gene of interest along with pGL3 Basic vector having promoter gene.
Every time I got results like 1.2 fold to 1.6 fold higher than control (Empty vector). Is this significant results? Either my gene of interest is binding to the promoter gene or not? or How can I increase the luciferase activity?
Kindly advice me to sort out this dilemma.
Thanks in advance.
Best Regards
It seems like your promoter is not strongly driving the expression of your gene. You could perhaps try confirming this by having a promoter like CMV or CAG in place. I think that should help your case.
More info would be helpful - your setup isn't clear. Are you looking for regulation of your promoter - indicated by the Luc readout generated by the promoter in the pGL3 - by the protein product of the 'vector harboring my gene of interest?' If so, you're in the right type of vector for the reporter. Is the empty vector a control?
Some things you might consider: Is your pGL3 generating a background of endogenously-driven luc - have you compared no-transfection to pGL3-transfection-alone? Is your transfection efficiency awesome (in those cell types it should be). Do you have a (+) control that would drive expression at your promoter without the use of the protein product generated by the other vector? Have you verified that the protein you think you're overexpressing actually is? Are you using a bright luciferase, and does its half-life make sense with the time course of your experiment? Have you optimized your cell density, and are you comparing similar cell densities across conditions? (You may want to jam a lot more cells in that well than you would ever use in a culture system, for instance - have you topped out how many cells you can squeeze into your well?!). Are you normalizing to cell number?
You need a statistical test to determine significance. Z-factor is one possibility; to find it you'll need readouts of multiple wells (at least 6) of each condition in *the same* experiment (n=1, all performed at the same time). 1.6-fold could be huge or nothing, depending on the variance in each condition.
Thanks AmirAli Bukhari.
Dear Melissa G Mendez thank you very much for detailed answer.
You are absolutely right. I want to check the binding of my protein of interest with promoter. Therefore, I inserted the promoter sequence in PGL3 basic vector, and gene coding my protein in another vector.
Yes I consider empty vector as a control.
No, I did not check nontransfection with PGL3 transfection alone.
Yes transfection efficiency is very good. Almost more than 80%.
I do not have positive control. I will try to manage positive control.
Yes I already checked protein integrity.
I am using Promega Luciferase assay kit.
I always seeded the cell with initial density 200000/ well of 12 wells plate, and transfected when cells confluency reached to 90%.
I shall stay in contact with you for further information.
Once again Thanks.
Best Regards
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