Dear Vania,

Normally, commercial available vectors contains CMW promotors. To me, it sounds like your problem is caused by promotor competition. You can easily check this by checking which promotor drives your proteins of interest in your constructs. If it is a CMW promotor and renilla and luciferase is also driven by a CMW promoter, then I'll put all my money on promotor competition being your problem.

In my experience, luciferase assays are not that good for the experiments you need to do. If you want to use luciferase, you should use a promotorless luciferase vector or a vector with a different kind of promotor than the promotor in your protein expressing vector. 

Regards

Frederik

Dear Frederik, 

Thanks for your time and help. We review our plasmids, we used three different plasmids with CMV or SV40 promoters. Renilla and Luciferase plasmids are the same promoter, so I don´t know where is the problem. We still have the same situation. 

Thanks again!

The purpose of the DLR assay is to check the activity of your promoter. If, in your case, the promoter is cloned into firefly containing vector such as pGL4, then you need to use the renilla-containing vector as the internal control. This is used to negate any problems caused by poor transfection and experimental variability.

Usually, the control plasmid is chosen with a weak promoter for its basal activity. Only in cases where the luciferase does not express a strong promoter is used.

In your case you need to perform the experiment with a promoterless internal control such as pGL4-basic. From your details it looks as if both SV40 and CMV promoters seem to be affected by your setup.

Subramaniyam

Subramaniyam, 

Thanks a lot for your time to answer my question, we have been working in the solution considering the acquisition of a promoterless of pGL4. Have a great day!!! =)