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Monitoring Intracellular pH Change with a Genetically Encoded and Ratiometric Luminescence Sensor in Yeast and Mammalian Cells

  • Yunfei Zhang
  • J. Brian Robertson
  • Qiguang Xie
  • Carl Hirschie JohnsonEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1461)

Abstract

“pHlash” is a novel bioluminescence-based pH sensor for measuring intracellular pH, which is developed based on Bioluminescence Resonance Energy Transfer (BRET). pHlash is a fusion protein between a mutant of Renilla luciferase (RLuc) and a Venus fluorophore. The spectral emission of purified pHlash protein exhibits pH dependence in vitro. When expressed in either yeast or mammalian cells, pHlash reports basal pH and cytosolic acidification. In this chapter, we describe an in vitro characterization of pHlash, and also in vivo assays including in yeast cells and in HeLa cells using pHlash as a cytoplasmic pH indicator.

Key words

pH BRET Luminescence Resonance transfer Ratiometric 

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Copyright information

© Springer Science+Business Media New York 2016

Authors and Affiliations

  • Yunfei Zhang
    • 1
    • 2
  • J. Brian Robertson
    • 1
    • 3
  • Qiguang Xie
    • 1
    • 4
  • Carl Hirschie Johnson
    • 1
    Email author
  1. 1.Department of Biological SciencesVanderbilt UniversityNashvilleUSA
  2. 2.Modern Experiment Technology CenterAnhui UniversityHefeiChina
  3. 3.Department of BiologyMiddle Tennessee State UniversityMurfreesboroUSA
  4. 4.Laboratory of Molecular and Cellular Biology, College of Life SciencesHebei Normal UniversityHebeiChina

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