Fluorescence cross-correlation spectroscopy (FCCS) is an advanced fluorescence technique that can quantify protein-protein interactions in vivo. Due to the dynamic, heterogeneous nature of the membrane, special considerations must be made to interpret FCCS data accurately. In this study, we describe a method to quantify the oligomerization of membrane proteins tagged with two commonly used fluorescent probes, mCherry (mCH) and enhanced green (eGFP) fluorescent proteins. A mathematical model is described that relates the relative cross-correlation value (fc) to the degree of oligomerization. This treatment accounts for mismatch in the confocal volumes, combinatoric effects of using two fluorescent probes, and the presence of non-fluorescent probes. Using this model, we calculate a ladder of fc values which can be used to determine the oligomer state of membrane proteins from live-cell experimental data. Additionally, a probabilistic mathematical simulation is described to resolve the affinity of different dimeric and oligomeric protein controls.

中文翻译：

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用荧光互相关光谱法量化膜蛋白寡聚化

荧光互相关光谱 (FCCS) 是一种先进的荧光技术，可以量化体内蛋白质 - 蛋白质相互作用。由于膜的动态、异质性，必须特别考虑以准确解释 FCCS 数据。在这项研究中，我们描述了一种量化标记有两种常用荧光探针、mCherry (mCH) 和增强型绿色 (eGFP) 荧光蛋白的膜蛋白寡聚化的方法。描述了将相对互相关值 (fc) 与低聚度相关联的数学模型。这种处理解释了共焦体积的不匹配、使用两个荧光探针的组合效应以及非荧光探针的存在。使用这个模型，我们计算了 fc 值的阶梯，可用于从活细胞实验数据中确定膜蛋白的寡聚体状态。此外，还描述了概率数学模拟来解决不同二聚体和寡聚体蛋白质对照的亲和力。