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

Abstract

Summary: 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 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Keywords

• Single Cell
• Cell-based Assays
• Microscopy
• Molecular/Chemical Probes
• Chemistry