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.
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Acknowledgments
Financial support was provided by the Signaling and Cell Cycle Regulation Training Grant (NIH T32 GM08759) to J.G.P. and NIH GM084027 and Alfred P. Sloan Fellowship to A.E.P.
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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. In: Zhang, J., Ni, Q., Newman, R. (eds) Fluorescent Protein-Based Biosensors. Methods in Molecular Biology, vol 1071. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-622-1_3
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DOI: https://doi.org/10.1007/978-1-62703-622-1_3
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