Institute of Multidisciplinary Research for Advanced Materials, Tohoku University, Sendai, Miyagi 980-8577, Japan.
Graduate School of Life Sciences, Tohoku University, Sendai, Miyagi 980-8577, Japan.
STAR Protoc. 2021 Mar 16;2(2):100395. doi: 10.1016/j.xpro.2021.100395. eCollection 2021 Jun 18.
Quantitative analysis using a turn-on fluorescent probe is inherently difficult due to the dependency of the fluorescence intensity on the probe concentration. To overcome this limitation, we developed an in situ quantification method using a turn-on fluorescent probe and a standard fluorophore, which are colocalized by protein tag technology. This protocol describes the synthesis of a Zn probe, named , and the procedure to quantify the labile Zn concentration in the Golgi of live HeLa cells by confocal fluorescence microscopy. For complete details on the use and execution of this protocol, please refer to Kowada et al. (2020).
由于荧光强度依赖于探针浓度，因此使用开-关型荧光探针进行定量分析本质上具有一定难度。为了克服这一限制，我们开发了一种使用开-关型荧光探针和标准荧光团的原位定量方法，该方法通过蛋白质标记技术实现共定位。本方案描述了 Zn 探针的合成，命名为 ，并描述了通过共聚焦荧光显微镜定量活 HeLa 细胞高尔基体中不稳定 Zn 浓度的过程。有关该方案的使用和执行的完整详细信息，请参见 Kowada 等人（2020 年）。
