Preparing monodisperse macromolecular samples for successful biological small-angle X-ray and neutron-scattering experiments.
Preparing monodisperse macromolecular samples for successful biological small-angle X-ray and neutron-scattering experiments.
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DOI：
10.1038/nprot.2016.113
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发表时间：
2016-11
期刊：
影响因子：
14.8
通讯作者：
Svergun DI
中科院分区：
文献类型：
--
作者：
Jeffries CM;Graewert MA;Blanchet CE;Langley DB;Whitten AE;Svergun DI
小角X射线和中子散射（SAXS和SANS）是用于提取结构参数并确定溶液中生物大分子，复合物和组装体的整体结构和形状的技术。从样品测量的散射强度包含来自照明样品体积内的所有原子的贡献，包括溶剂和缓冲液组分以及感兴趣的大分子。为了获得结构信息，必须制备完全匹配的溶剂空白，以便可以从样品散射中准确减去背景散射贡献，以获得样品中大分子的净散射。此外，由污染物、聚集体、不匹配的溶剂、辐射损伤或其他因素引起的样品异质性会严重影响数据分析并使其复杂化，因此在实验期间样品必须是纯的和单分散的。该协议概述了SAXS和SANS的基本物理原理，并揭示了该技术的基本概念原则如何最终“转化”为实际的实验室指导，用于生产足够高质量的散射实验样品。该程序描述了如何使用凝胶电泳、尺寸排阻色谱和光散射制备和表征SAXS和SANS的蛋白质和核酸样品。还包括特定于X射线（在线尺寸排阻色谱SAXS）和中子的程序，特别是准备用于对比度匹配/变化实验和蛋白质氘标记的样品。
Small-angle X-ray and neutron scattering (SAXS and SANS) are techniques used to extract structural parameters and determine the overall structures and shapes of biological macromolecules, complexes and assemblies in solution. The scattering intensities measured from a sample contain contributions from all atoms within the illuminated sample volume including the solvent and buffer components as well as the macromolecules of interest. In order to obtain structural information, it is essential to prepare an exactly matched solvent blank so that background scattering contributions can be accurately subtracted from the sample scattering to obtain the net scattering from the macromolecules in the sample. In addition, sample heterogeneity caused by contaminants, aggregates, mismatched solvents, radiation damage or other factors can severely influence and complicate data analysis so it is essential that the samples are pure and monodisperse for the duration of the experiment. This Protocol outlines the basic physics of SAXS and SANS and reveals how the underlying conceptual principles of the techniques ultimately ‘translate’ into practical laboratory guidance for the production of samples of sufficiently high quality for scattering experiments. The procedure describes how to prepare and characterize protein and nucleic acid samples for both SAXS and SANS using gel electrophoresis, size exclusion chromatography and light scattering. Also included are procedures specific to X-rays (in-line size exclusion chromatography SAXS) and neutrons, specifically preparing samples for contrast matching/variation experiments and deuterium labeling of proteins.