Luciferase Assay

[Alyaa Mahmoud]

Did anyone work with luciferase before ? We are trasnfecting HepG2 cells with Luc containning vectors and we are using "Victor" luminometer" to measure the signal but we are getting very irrelevant results and the negative control (GFP, which should give a zero signal" is displaying a good signal

Does anyone have any idea how this could be handled ?

Thanks

[ethidiumbromide]

It sounds to me like your transfection wasn't very good (with the luc). Can you increase the concentration of plasmid you are transfecting? The plasmid is either no good or there is too little of it. Could your purity be bad? How's the 260/280 for your plasmid?

[Alyaa Mahmoud]

Yeah it is possible to increase the concentration of the plasmid, but how do I know that this is the real problem ? I don;t understand what the 260/280 for a plasmid is ?

The major problem I am facing is that no one in my uni ever worked with luciferase before, so if you did, I would highly appreciate if you can email me with the optimized protocols you were using, the process of adjusting the Luminometer itself, constructing a standard curve or so...

Thanks a lot for your time
Have a great day

[ethidiumbromide]

I wish I could help, but I don't think I can give you the information you are looking for. Maybe you can do a google search for luciferase protocols. You should be able to find something.

I don't know if you made the plasmid yourself, or if it was store bought. Maybe the manufacturer has some information on their website, or with the product sheet that came with the plasmid. If you made it yourself, you would have needed to check the 260/280 to insure purity. The 260/280 is the O.D. of your plasmid at these two wavelengths, divided by each other. 260 is the wavelength at which DNA is measured. 280 is the wavelength most proteins absorb. The number is usually considered normal if it is between 1.8 and 2.0.

Good luck!

[kiki06]

Hello Alyaa, ethidiumbromide mentions some good points.

I have done dual luciferase assays with the Promega kit. Are you using a kit based method or home/lab made stuff?

Also make sure you set the properties of the luminometer carefully as it may not be picking up your signal. Its quite important to use the proper LUC buffers let us know what you are using and what settings you are using for the luminometer.

What may help you is to download and read the Promega dual luciferase assay kit manual as it has many of the same troubleshooting and instructions you will use.

See it here:[Only registered users see links. ]