Fig. 4

Change of cAMP-dependent Epac1-camps-His₆ fluorescence using desiccated and re-constituted samples. Purified Epac1-camps-His₆ protein was diluted to a concentration of 0.7 μM in IS buffer, transferred to all wells of a 96 MWP, and desiccated in a fridge. Protein in 48 wells was reconstituted in bi-destilled H₂O and protein in the remaining wells was reconstituted in TE-buffer. This design allowed simultaneous four-fold measurements. Excitation was at 430 nm and emission was recorded at 475 nm (ECFP) and 530 nm (EYFP). After recording the basal fluorescence for ECFP and EYFP, increasing concentrations of cAMP were added. The EYFP/ECFP ratio (R) for each well was calculated and normalized to the ratio of the basal fluorescence in the absence of cAMP (R₀). Normalized data (mean values ± SD) were plotted against cAMP concentrations. EC₅₀ values were calculated with a four-parameter nonlinear regression using GraphPad Prism v5.04. A representative of two independently performed experiments is shown