Transfection of nuclear receptor response element

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Katie
Katie's picture
Transfection of nuclear receptor response element

Hello, My lab is having great difficulty getting a PPRE luciferase reporter assay to work. I am able to get activation with ligand, but when we do anything else to manipulate the system, we are not getting any induction. This is very strange b/c typically we see coactivation with coactivators, etc. This system has completely stopped working for us. We have gotten new cells, new plasmids, new plasmid preps, new water, new media, you name, it, we've tried it, I think. It has been such a mystery as to why these luciferase assays are no longer showing induction as they were in the past. Any suggestions or ideas would be greatly appreciated.

Thanks

saswati1
saswati1's picture
You can certainly sequence it

You can certainly sequence it to check whether the plasmid is fine. You can request again to the person who you got the plasmid from. I used this plasmid and got it from Dr. Peter Tontonoz (UCLA).

Katie
Katie's picture
The problem isn't just one

The problem isn't just one reporter. We are having trouble with all of our reporters and even the gal4 systems. It's the strangest thing.

plasko20
plasko20's picture
 I think you should test your

 I think you should test your cells for mycoplasma. There are several kits available to do this by either a PCR method or cells staining method.  Even if you have new cells if its the same incubator the mycoplasma can re-infect these. Or perhaps it was in your TC hood, media, FBS etc. There are plenty sources of potential contamination. Mycoplasma screws up luciferase results. 
Another option is that the pH of your water, or something else has changed. 
Or perhaps your transfection reagent has gone off.