title: Monitoring Intracellular pH Change with a Genetically Encoded and Ratiometric Luminescence Sensor in Yeast and Mammalian Cells.
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.
authors.affiliation: Department of Biological Sciences, Vanderbilt University, Nashville, TN, 37235, USA.|Modern Experiment Technology Center, Anhui University, Hefei, Anhui, 230601, China.
authors.forename: Yunfei
authors.initials: Y
authors.lastname: Zhang
authors.affiliation: Department of Biological Sciences, Vanderbilt University, Nashville, TN, 37235, USA.|Department of Biology, Middle Tennessee State University, Murfreesboro, TN, USA.
authors.forename: J Brian
authors.initials: JB
authors.lastname: Robertson
authors.affiliation: Department of Biological Sciences, Vanderbilt University, Nashville, TN, 37235, USA.|Laboratory of Molecular and Cellular Biology, College of Life Sciences, Hebei Normal University, Hebei, China.
authors.forename: Qiguang
authors.initials: Q
authors.lastname: Xie
authors.affiliation: Department of Biological Sciences, Vanderbilt University, Nashville, TN, 37235, USA. carl.h.johnson@vanderbilt.edu.
authors.forename: Carl Hirschie
authors.initials: CH
authors.lastname: Johnson
mesh_terms: D000818:Animals; D015374:Biosensing Techniques; D020148:Butyric Acid / Q000737:chemistry; D002460:Cell Line; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods; D015870:Gene Expression; D006367:HeLa Cells; D006801:Humans; D006863:Hydrogen-Ion Concentration; D042541:Intracellular Space; D008163:Luminescent Measurements / Q000379:methods*; D008164:Luminescent Proteins / Q000235:genetics* / Q000378:metabolism*; D057054:Molecular Imaging / Q000379:methods; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism
chemical_list: D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D020148:Butyric Acid
keywords: BRET; Luminescence; Ratiometric; Resonance transfer; pH