Protocol for measuring labile cytosolic Zn2+ using an in situ calibration of a genetically encoded FRET sensor

  • STAR Protoc. 2024 Jun 21;5(2):103130. doi: 10.1016/j.xpro.2024.103130.
Samuel E Holtzen  1 ,  Ananya Rakshit  2 ,  Amy E Palmer  3
Affiliations
  • 1. BioFrontiers Institute and Department of Biochemistry, 3415 Colorado Avenue, University of Colorado Boulder, Boulder, CO 80303, USA; Department of Molecular Cellular Developmental Biology and BioFrontiers Institute, University of Colorado Boulder, Boulder, CO 80309, USA.
  • 2. BioFrontiers Institute and Department of Biochemistry, 3415 Colorado Avenue, University of Colorado Boulder, Boulder, CO 80303, USA.
  • 3. BioFrontiers Institute and Department of Biochemistry, 3415 Colorado Avenue, University of Colorado Boulder, Boulder, CO 80303, USA. Electronic address: [email protected].
PMID: 38870018 DOI: 10.1016/j.xpro.2024.103130
Abstract

Zinc (Zn2+) plays roles in structure, catalysis, and signaling. The majority of cellular Zn2+ is bound by proteins, but a fraction of total Zn2+ exists in a labile form. Here, we present a protocol for measuring labile cytosolic Zn2+ using an in situ calibration of a genetically encoded Förster resonance energy transfer (FRET) sensor. We describe steps for producing buffered Zn2+ solutions for performing an imaging-based calibration and analyzing the imaging data generated to determine labile Zn2+ concentration in single cells. For complete details on the use and execution of this protocol, please refer to Rakshit and Holtzen et al.1.

Keywords
Cell-based Assays; Chemistry; Microscopy; Molecular/Chemical Probes; Single Cell.
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