测量基因编码钙传感器的原位解离常数（Kd）
Measuring the in situ Kd of a genetically encoded Ca2+ sensor.
作者信息
Park J Genevieve, Palmer Amy E
机构信息
Department of Chemistry and Biochemistry and BioFrontiers Institute, University of Colorado, Boulder, Colorado 80309.
出版信息
Cold Spring Harb Protoc. 2015 Jan 5;2015(1):pdb.prot076554. doi: 10.1101/pdb.prot076554.
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.
在过去十年中，使用基因编码的钙离子传感器（GECIs）对细胞内钙离子进行长期监测已变得越来越普遍。发射比率型GECIs，如黄色变色龙家族中的那些，可以用于进行定量测量，这意味着它们的荧光信号可以转换为游离钙离子浓度（[Ca2+]游离）。这种转换的准确性仅取决于传感器对钙离子的表观解离常数（K'd），而K'd又取决于温度、pH值和盐浓度。本方案描述了一种在活细胞（原位）中对胞质、核或线粒体传感器进行滴定的方法。
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