英国伯明翰大学Dirk-Peter Herten团队开发出一种用于染色优化和分子计数的通用标记程度测定方法。2024年8月8日，国际知名学术期刊《自然—方法学》在线发表了这一成果。

研究人员介绍了一种名为蛋白标签标记程度（ProDOL）的标记程度（DOL）测定技术，它可以快速量化和优化蛋白标签标记。通过ProDOL，可以轻松评估影响标记效率的各种因素，包括底物类型、孵育时间和浓度，以及样品固定和细胞类型。

研究人员应用ProDOL揭示了人类免疫缺陷病毒-1致病因子Nef如何调节CD4 T细胞激活，测量了信号微簇中适配蛋白SLP-76的总拷贝数和激活拷贝数。ProDOL被证明是一种多功能且稳健的标记校准工具，能够确定标记效率、优化策略和量化蛋白量子化学计量。

研究人员表示，确定标记与靶标比率，也称为DOL，对定量荧光显微镜至关重要。高DOL且最小化非特异性标记对荧光显微镜有利。然而，目前还没有稳健、通用且易于使用的工具来测量细胞特异性标记效率。

附：英文原文

Title: ProDOL: a general method to determine the degree of labeling for staining optimization and molecular counting

Author: Tashev, Stanimir Asenov, Euchner, Jonas, Yserentant, Klaus, Hnselmann, Siegfried, Hild, Felix, Chmielewicz, Wioleta, Hummert, Johan, Schwrer, Florian, Tsopoulidis, Nikolaos, Germer, Stefan, Samannshausen, Zoe, Fackler, Oliver T., Klingmller, Ursula, Herten, Dirk-Peter

Issue&Volume: 2024-08-08

Abstract: Determining the label to target ratio, also known as the degree of labeling (DOL), is crucial for quantitative fluorescence microscopy and a high DOL with minimal unspecific labeling is beneficial for fluorescence microscopy in general. Yet robust, versatile and easy-to-use tools for measuring cell-specific labeling efficiencies are not available. Here we present a DOL determination technique named protein-tag DOL (ProDOL), which enables fast quantification and optimization of protein-tag labeling. With ProDOL various factors affecting labeling efficiency, including substrate type, incubation time and concentration, as well as sample fixation and cell type can be easily assessed. We applied ProDOL to investigate how human immunodeficiency virus-1 pathogenesis factor Nef modulates CD4 T cell activation measuring total and activated copy numbers of the adapter protein SLP-76 in signaling microclusters. ProDOL proved to be a versatile and robust tool for labeling calibration, enabling determination of labeling efficiencies, optimization of strategies and quantification of protein stoichiometry.

DOI: 10.1038/s41592-024-02376-6

Source: https://www.nature.com/articles/s41592-024-02376-6