Fig. 1
From: Establishing a sensitive fluorescence-based quantification method for cyclic nucleotides
Characterization of Epac1-camps-His6 in multi-well plates. Purified Epac1-camps-His6 protein was diluted to a concentration of 0.7 μM in IS buffer and 90 μl of the sample was added to each well of a 96 multi well plate. 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 or cGMP were added to each well. The final volume was 100 μl and final concentrations of cyclic nucleotides ranged from 10− 9 - 10− 4 M. The EYFP/ECFP ratio (R) for each well was calculated and normalized to the ratio of the basal fluorescence in the absence of cyclic nucleotides (R0). Normalized data were plotted against cyclic nucleotide concentrations. Each data point is given as mean value (±SD) from four identically treated wells of a representative experiment. EC50 values were calculated with a four-parameter nonlinear regression using GraphPad Prism v5.04