title: Measuring the in situ Kd of a genetically encoded Ca2+ sensor.
abstract: The use of genetically encoded Ca(2+) sensors (GECIs) for long-term monitoring of intracellular Ca(2+) has become increasingly common in the last decade. Emission-ratiometric GECIs, such as those in the Yellow Cameleon family, can be used to make quantitative measurements, meaning that their fluorescence signals can be converted to free Ca(2+) concentrations ([Ca(2+)]free). This conversion is only as accurate as the sensor's apparent dissociation constant for Ca(2+) (K'd), which depends on temperature, pH, and salt concentration. This protocol describes a method for performing a titration, in living cells (in situ), of cytosolic, nuclear, or mitochondrial sensors.
authors.affiliation: Department of Chemistry and Biochemistry and BioFrontiers Institute, University of Colorado, Boulder, Colorado 80309.
authors.forename: J Genevieve
authors.initials: JG
authors.lastname: Park
authors.affiliation: Department of Chemistry and Biochemistry and BioFrontiers Institute, University of Colorado, Boulder, Colorado 80309.
authors.forename: Amy E
authors.initials: AE
authors.lastname: Palmer
mesh_terms: D000818:Animals; D015374:Biosensing Techniques / Q000379:methods*; D002118:Calcium / Q000378:metabolism*; D002478:Cells, Cultured; D002614:Chelating Agents / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D031541:Fluorescence Resonance Energy Transfer; D006801:Humans; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism*; D061848:Optical Imaging; D011485:Protein Binding; D014162:Transfection
chemical_list: D002614:Chelating Agents; D008164:Luminescent Proteins; D002118:Calcium