使用基因编码的 FRET 传感器进行原位校准来测量细胞溶质可动锌的方案。
Protocol for measuring labile cytosolic Zn using an in situ calibration of a genetically encoded FRET sensor.
机构信息
BioFrontiers Institute and Department of Biochemistry, 3415 Colorado Avenue, University of Colorado Boulder, Boulder, CO 80303, USA; Department of Molecular Cellular Developmental Biology and BioFrontiers Institute, University of Colorado Boulder, Boulder, CO 80309, USA.
BioFrontiers Institute and Department of Biochemistry, 3415 Colorado Avenue, University of Colorado Boulder, Boulder, CO 80303, USA.
出版信息
STAR Protoc. 2024 Jun 21;5(2):103130. doi: 10.1016/j.xpro.2024.103130. Epub 2024 Jun 12.
Zinc (Zn) plays roles in structure, catalysis, and signaling. The majority of cellular Zn is bound by proteins, but a fraction of total Zn exists in a labile form. Here, we present a protocol for measuring labile cytosolic Zn using an in situ calibration of a genetically encoded Förster resonance energy transfer (FRET) sensor. We describe steps for producing buffered Zn solutions for performing an imaging-based calibration and analyzing the imaging data generated to determine labile Zn concentration in single cells. For complete details on the use and execution of this protocol, please refer to Rakshit and Holtzen et al..
锌（Zn）在结构、催化和信号转导中发挥作用。大多数细胞内的锌与蛋白质结合，但一部分总锌以可移动的形式存在。在这里，我们提出了一种使用遗传编码的Förster 共振能量转移（FRET）传感器进行原位校准来测量细胞溶质中可移动锌的方案。我们描述了用于进行基于成像的校准以及分析生成的成像数据的缓冲锌溶液的制备步骤，以确定单细胞中可移动锌的浓度。有关该方案的使用和执行的完整详细信息，请参考 Rakshit 和 Holtzen 等人的研究。
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