Abstract
Cyclic ADP-ribose (cADPR) is an intracellular messenger that triggers the release of calcium ions from intracellular stores in a variety of cell types. The fluorometric cycling assay has become the preferred method for measuring cADPR due to its high level of sensitivity (in the sub-nanomolar range) and its use of commercially available reagents. Additionally, the assay is performed in multiwell plates, making it suitable for high throughput screening using a fluorescence plate reader. The findings reported in this paper present several problems that may be encountered during various stages of the assay, and provide solutions to these problems. Modifications to the assay address reduced recovery of sample and cADPR with removal of perchloric acid (PCA) using organic solvent, reduction in diaphorase activity with heat treatment, and effects on resorufin fluorescence by pH range. Using these modifications, we report an increase of approximately 15% in recovery of brain cADPR, and show that between-subject variability is greatly reduced. We hope that these observations will encourage more widespread application of this valuable assay.
Keywords: Cyclic ADP-ribose, cycling assay, ADP-ribosyl cyclase, pyridine nucleotides, calcium signalling, NAD+, NADH
Combinatorial Chemistry & High Throughput Screening
Title: Modifications to Increase the Efficiency of the Fluorometric Cycling Assay for Cyclic ADP-Ribose
Volume: 9 Issue: 8
Author(s): Genevieve S. Young and James B. Kirkland
Affiliation:
Keywords: Cyclic ADP-ribose, cycling assay, ADP-ribosyl cyclase, pyridine nucleotides, calcium signalling, NAD+, NADH
Abstract: Cyclic ADP-ribose (cADPR) is an intracellular messenger that triggers the release of calcium ions from intracellular stores in a variety of cell types. The fluorometric cycling assay has become the preferred method for measuring cADPR due to its high level of sensitivity (in the sub-nanomolar range) and its use of commercially available reagents. Additionally, the assay is performed in multiwell plates, making it suitable for high throughput screening using a fluorescence plate reader. The findings reported in this paper present several problems that may be encountered during various stages of the assay, and provide solutions to these problems. Modifications to the assay address reduced recovery of sample and cADPR with removal of perchloric acid (PCA) using organic solvent, reduction in diaphorase activity with heat treatment, and effects on resorufin fluorescence by pH range. Using these modifications, we report an increase of approximately 15% in recovery of brain cADPR, and show that between-subject variability is greatly reduced. We hope that these observations will encourage more widespread application of this valuable assay.
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Cite this article as:
Young S. Genevieve and Kirkland B. James, Modifications to Increase the Efficiency of the Fluorometric Cycling Assay for Cyclic ADP-Ribose, Combinatorial Chemistry & High Throughput Screening 2006; 9 (8) . https://dx.doi.org/10.2174/138620706778249749
DOI https://dx.doi.org/10.2174/138620706778249749 |
Print ISSN 1386-2073 |
Publisher Name Bentham Science Publisher |
Online ISSN 1875-5402 |
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