Transcreener® assays detect ADP, AMP/GMP, UDP and GDP nucleotides. These di- and monophosphate nucleotides form during enzymatic reactions and therefore report on the activity of methyltransferases, kinases, GTPases, phosphodiesterases and others. Apart from fluorescence and TR-FRET assays, a fluorescence polarization assays are available. The respective analyte binds to the analyte-specific antibody, thereby displacing a fluorescent structurally related tracer that is bound to the antibody initially. The tracer then freely rotates leading to depolarization of emission light and reduction of the FP value.
This application note presents the ADP, GDP and UDP FP assays read on a PHERAstar® microplate reader. The assays exhibited Z' values >0.8 at 10 % nucleotide conversion proving the robustness of the assay. Moreover, the ability of the PHERAstar to simultaneously detect the two emissions required for FP made the measurement exceptionally fast.