Methods for using a genetically encoded fluorescent biosensor to monitor nuclear NAD +

Michael S. Cohen, Melissa L. Stewart, Richard H. Goodman, Xiaolu A. Cambronne

Research output: Chapter in Book/Report/Conference proceedingChapter

3 Scopus citations

Abstract

Free nicotinamide adenine dinucleotide (NAD + ) serves as substrate for NAD + -consuming enzymes. As such, the local concentration of free NAD + can influence enzymatic activities. Here we describe methods for using a fluorescent, genetically-encoded sensor to measure subcellular NAD + concentrations. We also include a discussion of the limitations and potential applications for the current sensor. Presented in this chapter are (1) guidelines for calibrating instrumentation and experimental setups using a bead-based method, (2) instructions for incorporating required controls and properly performing ratiometric measurements in cells, and (3) descriptions of how to evaluate relative and quantitative fluctuations using appropriate statistical methods for ratio-of-ratio measurements.

Original languageEnglish (US)
Title of host publicationMethods in Molecular Biology
PublisherHumana Press Inc.
Pages391-414
Number of pages24
DOIs
StatePublished - 2018

Publication series

NameMethods in Molecular Biology
Volume1813
ISSN (Print)1064-3745

Keywords

  • ARTD
  • Biosensor
  • Circularly permuted fluorescent protein
  • Fluorescent sensor
  • Metabolite
  • NAD
  • Nicotinamide adenine dinucleotide
  • PARP

ASJC Scopus subject areas

  • Molecular Biology
  • Genetics

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    Cohen, M. S., Stewart, M. L., Goodman, R. H., & Cambronne, X. A. (2018). Methods for using a genetically encoded fluorescent biosensor to monitor nuclear NAD + In Methods in Molecular Biology (pp. 391-414). (Methods in Molecular Biology; Vol. 1813). Humana Press Inc.. https://doi.org/10.1007/978-1-4939-8588-3_26