Sedimentation velocity (SV) analytical ultracentrifugation (AUC) is a classic technique for the real-time observation of free macromolecular migration in solution driven by centrifugal force. This enables the analysis of macromolecular mass, shape, size distribution, and interactions. Although traditionally limited to determination of the sedimentation coefficient and binding affinity of proteins in the micromolar range, the implementation of modern detection and data analysis techniques has resulted in marked improvements in detection sensitivity and size resolution during the past decades. Fluorescence optical detection now permits the detection of recombinant proteins with fluorescence excitation at 488 or 561 nm at low picomolar concentrations, allowing for the study of high-affinity protein self-association and hetero-association. Compared with other popular techniques for measuring high-affinity protein-protein interactions, such as biosensing or calorimetry, the high size resolution of complexes at picomolar concentrations obtained with SV offers a distinct advantage in sensitivity and flexibility of the application. Here, we present a basic protocol for carrying out fluorescence-detected SV experiments and the determination of the size distribution and affinity of protein-antibody complexes with picomolar K_D values. Using an EGFP-nanobody interaction as a model, this protocol describes sample preparation, ultracentrifugation, data acquisition, and data analysis. A variation of the protocol applying traditional absorbance or an interference optical system can be used for protein-protein interactions in the micromolar K_D value range. Sedimentation experiments typically take ∼3 h of preparation and 6-12 h of run time, followed by data analysis (typically taking 1-3 h).

中文翻译：

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使用荧光检测的沉降速度来研究高亲和力蛋白质相互作用。

沉降速度（SV）分析超速离心（AUC）是实时观察溶液中由离心力驱动的自由大分子迁移的经典技术。这使得能够分析大分子质量、形状、尺寸分布和相互作用。尽管传统上仅限于测定微摩尔范围内蛋白质的沉降系数和结合亲和力，但现代检测和数据分析技术的实施在过去几十年中显着提高了检测灵敏度和尺寸分辨率。荧光光学检测现在可以在低皮摩尔浓度下在 488 或 561 nm 处进行荧光激发来检测重组蛋白，从而可以研究高亲和力蛋白自缔合和异源缔合。与测量高亲和力蛋白质-蛋白质相互作用的其他流行技术（例如生物传感或量热法）相比，使用 SV 获得的皮摩尔浓度复合物的高尺寸分辨率在应用的灵敏度和灵活性方面具有明显的优势。在这里，我们提出了一个基本方案，用于进行荧光检测 SV 实验，并确定具有皮摩尔 KD 值的蛋白质-抗体复合物的尺寸分布和_亲和力。该协议使用 EGFP-纳米抗体相互作用作为模型，描述了样品制备、超速离心、数据采集和数据分析。应用传统吸光度或干涉光学系统的方案的变体可用于微摩尔 KD 值范围内的蛋白质-蛋白质_相互作用。沉降实验通常需要 ∼3 小时的准备时间和 6-12 小时的运行时间，然后进行数据分析（通常需要 1-3 小时）。