Nomura Y, Tanaka H, Poellinger L, Higashino F, Kinjo M
Laboratory of Supramolecular Biophysics, Research Institute for Electronic Science, Hokkaido University, Sapporo, Japan.
Cytometry. 2001 May 1;44(1):1-6.
Because the process of protein translation is an event of sparse molecules, the measurement requires high sensitivity. One of the candidates for studying the molecules is fluorescence correlation spectroscopy (FCS), which gleans quantitative information from fluctuating fluorescence signals in a diluted solution.
Using FCS, the translation products of expression plasmid for green fluorescent protein (GFP) and its fusion proteins were measured in vitro and in vivo.
In in vitro translation, the number of products increased linearly for 90 min upon concentration of the plasmid. The autocorrelation function for GFP was fitted with a one-component model with a diffusion time of 0.18 ms, which was identical to the value expected from the molecular weight. In the cases of GFP- tagged hypoxia-inducible factor-1 alpha and glucocorticoid receptor, each fitting result was significantly improved with a two-component model. The slow component with a diffusion time of 6 ms appeared to be related to the ribosome or polysome. In response to the addition of dexamethasone, the nuclear translocation from cytosol clearly induced the decrease in number of molecules in the focal point.
FCS permits monitoring of the number of molecules translated in vitro and in vivo, the translation rate, and the molecular weight.
由于蛋白质翻译过程是一个涉及稀少分子的事件，该测量需要高灵敏度。用于研究这些分子的候选方法之一是荧光相关光谱法（FCS），它从稀释溶液中波动的荧光信号中获取定量信息。
使用FCS，在体外和体内测量绿色荧光蛋白（GFP）表达质粒及其融合蛋白的翻译产物。
在体外翻译中，随着质粒浓度增加，产物数量在90分钟内呈线性增加。GFP的自相关函数用扩散时间为0.18毫秒的单组分模型拟合，这与根据分子量预期的值相同。在GFP标记的缺氧诱导因子-1α和糖皮质激素受体的情况下，用双组分模型拟合每个结果都有显著改善。扩散时间为6毫秒的慢组分似乎与核糖体或多核糖体有关。响应地塞米松的添加，从细胞质到细胞核的转位明显导致焦点处分子数量减少。
FCS能够监测体外和体内翻译的分子数量、翻译速率和分子量。
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