Hi,

I'm using a Cisbio Gs-coupled cAMP second messenger assay kit to screen test compounds against a Gi-coupled receptor. To do this, I activate the cells I'm working with using forskolin (increases cAMP release via adenylyl cyclase), then inhibit that response with compounds that activate the receptor (see image).

To plot dose-response curves of the compounds, the mean of the minimum response control wells (forskolin only) is converted to '0% activation', and the mean of maximum response control wells (forskolin + potent reference compound) is converted to '100% activation'. Unfortunately, my baseline response for the dose response curves (forskolin only) is very variable (see image).

I'm achieving Z factors of 0.5-0.6 which are reasonable, the curves are just very poor...

Does anyone have experience with Gi-coupled receptor screening assays? Have you had an issue like this before and have you been able to fix it? Any help would be appreciated!