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Methods for Using a Genetically Encoded Fluorescent Biosensor to Monitor Nuclear NAD+

  • Michael S. Cohen
  • Melissa L. Stewart
  • Richard H. Goodman
  • Xiaolu A. Cambronne
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1813)

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.

Key words

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

Notes

Acknowledgments

This work was supported by funding from the Hillcrest Committee Pilot Project Award and NIH DP2GM126897 and P30CA069533 to X.A.C. and The Pew Charitable Trusts to M.S.C. NAD+ sensors which are available to the academic research community under a material transfer agreement with Oregon Health & Science University (OHSU). The authors are listed as inventors on patent PCT/US15/62003 for the NAD+ sensor.

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Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2018

Authors and Affiliations

  • Michael S. Cohen
    • 1
  • Melissa L. Stewart
    • 2
  • Richard H. Goodman
    • 2
  • Xiaolu A. Cambronne
    • 2
    • 3
  1. 1.Department of Physiology and Pharmacology, Program in Chemical BiologyOregon Health and Science UniversityPortlandUSA
  2. 2.Vollum InstituteOregon Health and Science UniversityPortlandUSA
  3. 3.Department of Molecular BiosciencesUniversity of Texas at AustinAustinUSA

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