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Quantitative measurement of Ca2+ and Zn2+ in mammalian cells using genetically encoded fluorescent biosensors

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Abstract

Genetically encoded, ratiometric, fluorescent biosensors can be used to quantitatively measure intracellular ion concentrations in living cells. We describe important factors to consider when selecting a Ca2+ or Zn2+ biosensor, such as the sensor's dissociation constant (K d′) and its dynamic range. We also discuss the limits of quantitative measurement using these sensors and reasons why a sensor may perform differently in different biological systems or subcellular compartments. We outline protocols for (1) quickly confirming sensor functionality in a new biological system, (2) calibrating a sensor to convert a sensor's FRET ratio to ion concentration, and (3) titrating a sensor in living cells to obtain its K d′ under different experimental conditions. © 2014 Springer Science+Business Media, LLC.

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Park, J. G., & Palmer, A. E. (2014). Quantitative measurement of Ca2+ and Zn2+ in mammalian cells using genetically encoded fluorescent biosensors. Methods in Molecular Biology, 1071, 29–47. https://doi.org/10.1007/978-1-62703-622-1_3

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