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Fluorescence polarization competition experiment. WT MrkH was added to the reaction tube containing 1 nM F-c-di-GMP, resulting in a binding curve (MrkH was added to a final concentration of 10 μM). The Kd for binding was determined as 113 ± 19 nM. Then, 10 μM nonfluoresceinated c-di-GMP was added (arrow), which resulted in apparent competition with the fluoresceinated c-di-GMP as revealed by the decrease in millipolarization units to 0.
