Related Experiment Video
Updated: Jun 2, 2026
Measuring Nucleotide Binding to Intact, Functional Membrane Proteins in Real Time
Published on: March 11, 2021
High-throughput, homogeneous, fluorescence intensity-based measurement of adenosine diphosphate and other
Adam B Shapiro1, Laurel Hajec, Ning Gao
1Bioscience Department, Infection Innovative Medicines, AstraZeneca R&D Boston, Waltham, MA 02451, USA. adam.shapiro@astrazeneca.com
Abstract:
A new, homogeneous, high-throughput-compatible assay method is described for the fluorescence-based quantitation of nanomolar concentrations of ribonucleoside diphosphates (rNDPs). The principle of the method is the conversion of the rNDPs to RNA by the enzyme polynucleotide phosphorylase (EC 2.7.7.8) and detection of the RNA by the increased fluorescence of a commercial nucleic acid detection dye. A commercial RNA homopolymer complementary to the RNA product is included to increase the sensitivity for ADP and UDP. Standard curves for nanomolar concentrations of ADP, UDP, GDP, and CDP are shown. The assay detected 75 nM ADP produced by the pyruvate kinase-catalyzed phosphorylation of pyruvate with a signal-to-baseline ratio of 2.8. The assay may be used in either a continuous or a discontinuous mode.
