Example 40
Two fold serial dilutions of NADH, NADPH, NAD, and NADP (Sigma) were made in PBS starting from 0.313 uM. 10 ul of each dilution was transferred into wells of a 384-well plate. Detection reagents were made by adding 10 U/ml of rat diaphorase (Sigma), 40 uM proluciferin substrate PBI 4312 and NADP or NAD dependent enzyme amplification systems consisting of Glucose-6Phosphate Dehydrogenase (5 U/ml) and glucose-6P (0.5 mM) for NADP or Lactate Dehydrogenase (5 U/ml) and Lactate (40 mM) for NAD into Luciferin Detection Reagent (LDR; Promega Cat. No V8920). 10 ul of appropriate detection reagent was added to the dinucleotide samples. The reactions were incubated for 30 minutes at room temperature, and luminescence measured on a Tecan plate luminometer.
The results show that when the detection method described herein is combined with the dinucleotide specific amplification enzyme system, light is generated only in the samples containing appropriate dinucleotide (