Use of fluorescence-detected sedimentation velocity to study high-affinity protein interactions.
Use of fluorescence-detected sedimentation velocity to study high-affinity protein interactions.
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DOI：
10.1038/nprot.2017.064
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发表时间：
2017-09
期刊：
影响因子：
14.8
通讯作者：
Schuck P
中科院分区：
文献类型：
--
作者：
Chaturvedi SK;Ma J;Zhao H;Schuck P
沉降速度（SV）分析超离心是一种经典的技术，用于实时观察大分子在离心力驱动下在溶液中的自由迁移。这使得能够分析大分子质量、形状、尺寸分布和相互作用。虽然传统上仅限于确定蛋白质的沉降系数和结合亲和力在微摩尔范围内，现代检测和数据分析技术的实施导致了显着的改善，在过去的几十年中，在检测灵敏度和尺寸分辨率。荧光光学检测现在允许在488或561 nm处分别以低皮摩尔浓度用荧光激发检测重组蛋白，从而允许研究高亲和力蛋白质自缔合和异缔合。与用于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，用SV获得的皮摩尔浓度的复合物的高尺寸分辨率在灵敏度和应用灵活性方面提供了明显的优势。在这里，我们提出了一个基本的协议进行荧光检测沉降速度实验和测定的大小分布和亲和力的蛋白质-抗体复合物与皮摩尔KD。使用EGFP-纳米抗体相互作用作为模型，该协议描述了样品制备，超离心，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范围内的蛋白质-蛋白质相互作用。沉淀实验通常需要约3小时的准备和6-12小时的运行时间，然后通常需要1-3小时的数据分析。该协议描述了如何使用沉降速度（SV）分析超离心结合荧光光学检测的质量，形状，尺寸分布和相互作用的蛋白质的结合常数的分析。荧光检测沉降速度
Sedimentation velocity (SV) analytical ultracentrifugation is a classical technique for the real-time observation of macromolecular migration free in solution driven by centrifugal force. This enables the analysis of macromolecular mass, shape, size-distribution, and interactions. While traditionally limited to determine the sedimentation coefficient and binding affinity of proteins in the micromolar range, the implementation of modern detection and data analysis techniques has resulted in dramatic improvements in detection sensitivity and size-resolution during the last decades. Fluorescence optical detection now permits the detection of recombinant proteins with fluorescence excitation at 488 or 561 nm, respectively, at low picomolar concentrations, allowing for the study of high-affinity protein self-association and hetero-association. Compared to 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 application. Here we present a basic protocol for carrying out fluorescence-detected sedimentation velocity experiments and the determination of the size-distribution and affinity of protein-antibody complexes with picomolar KD. 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 interference optical system can be used for protein-protein interactions in the micromolar KD range. Sedimentation experiments typically take ~3 hours of preparation and 6—12 hours run time, followed by data analysis of usually 1—3 hours. This protocol describes how to use sedimentation velocity (SV) analytical ultracentrifugation in combination with fluorescence optical detection for the analysis of mass, shape, size-distribution, and binding constants of interacting proteins. Fluorescence detected sedimentation velocity
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影响因子：
7.4
作者：
Zhao, Huaying;Ma, Jia;Ingaramo, Maria;Andrade, Eric;MacDonald, Jeff;Ramsay, Glen;Piszczek, Grzegorz;Patterson, George H.;Schuck, Peter
通讯作者：
Schuck, Peter
影响因子：
64.8
作者：
Gavin, AC;Bösche, M;Superti-Furga, G
通讯作者：
Superti-Furga, G
影响因子：
3.4
作者：
Chaturvedi, Sumit K.;Zhao, Huaying;Schuck, Peter
通讯作者：
Schuck, Peter
影响因子：
4.8
作者：
Kingsbury, Jonathan S.;Laue, Thomas M.;Connors, Lawreen H.
通讯作者：
Connors, Lawreen H.
影响因子：
2.9
作者：
Husain B;Mukerji I;Cole JL
通讯作者：
Cole JL