I am having some difficulties with my luciferase reporter gene assays. The cells are being transfected with peptides that are dissolved in 25% acetic acid (stock). The final acetic acid concentration (media etc.) goes as low as 0.125% and still, this is interfering with the reporter gene assay. The peptides need to be dissolved in acid, and diluting them further isn't possible. Also, even at the 0.125% concentration of acid there is still no activity at the promoter.
Has anyone ever experienced this, or know what might help overcome it?
Thank you!
Hi,
What exactly are you measuring and in which system? Can you describe it more specifically? Are you using for example one of cell lines from invivogen, which means that you are measuring the luciferase activity in QuantiLuc medium?
We are measuring activity at the luciferase promoter which is measured using a luminometer and normalised to Renilla values. The cells are just HEKs and HeLas .
We transfect the cells with plasmids containing the gene of interest upstream of the luciferase promoter. The more luciferase activity, the higher the reading on the luminometer. We use a dual reporter assay system (purchased commercially)
They don't go into DMSO well and it interferes with the peptides entering the cell
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