High-throughput, homogeneous, fluorescence intensity-based measurement of adenosine diphosphate and other ribonucleoside diphosphates with nanomolar sensitivity |
| |
Authors: | Shapiro Adam B Hajec Laurel Gao Ning |
| |
Affiliation: | Bioscience Department, Infection Innovative Medicines, AstraZeneca R&D Boston, 35 Gatehouse Drive, Waltham, MA 02451, USA |
| |
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. |
| |
Keywords: | ADP UDP GDP CDP High throughput Assay Polynucleotide phosphorylase Pyruvate kinase |
本文献已被 ScienceDirect PubMed 等数据库收录! |
|