Characterising protein/detergent complexes by triple-detection size-exclusion chromatography.
Characterising protein/detergent complexes by triple-detection size-exclusion chromatography.
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DOI：
10.1186/s12575-015-0031-9
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发表时间：
2016
影响因子：
6.4
通讯作者：
Keller S
中科院分区：
文献类型：
--
作者：
Gimpl K;Klement J;Keller S
膜蛋白的体外研究通常依赖于用于蛋白溶解和稳定的去污剂。与膜蛋白结合的去污剂的量与成功的实验设计和数据分析相关，但通常是未知的。三重检测尺寸排阻色谱能够同时分离蛋白质/洗涤剂复合物和无蛋白质的洗涤剂胶束，并以直接和绝对的方式测定其摩尔质量。尺寸排阻色谱法用于分离不同的物质，而紫外吸光度，静态光散射和折射率测量允许蛋白质和洗涤剂组分的摩尔质量测定。我们完善了标准的实验和数据分析程序，以挑战逃避常规方法的膜蛋白样品。一般程序，包括准备步骤，测量和数据分析的常规和复杂的样品在困难的溶剂，如浓缩变性剂溶液的表征证明。该协议的适用性，但它的局限性和可能的解决方案进行了讨论，并提供了广泛的故障排除部分。我们建立并验证了三重检测尺寸排阻色谱的方案，使没有经验的用户能够执行和分析性能良好的蛋白质/去污剂复合物的测量。更有经验的用户提供了一个更复杂的分析程序的例子，允许在具有挑战性的分离条件下进行质量测定。
In vitro investigations of membrane proteins usually depend on detergents for protein solubilisation and stabilisation. The amount of detergent bound to a membrane protein is relevant to successful experiment design and data analysis but is often unknown. Triple-detection size-exclusion chromatography enables simultaneous separation of protein/detergent complexes and protein-free detergent micelles and determination of their molar masses in a straightforward and absolute manner. Size-exclusion chromatography is used to separate different species, while ultraviolet absorbance, static light scattering, and refractive index measurements allow molar mass determination of protein and detergent components. We refined standard experimental and data-analysis procedures for challenging membrane-protein samples that elude routine approaches. The general procedures including preparatory steps, measurements, and data analysis for the characterisation of both routine and complex samples in difficult solvents such as concentrated denaturant solutions are demonstrated. The applicability of the protocol but also its limitations and possible solutions are discussed, and an extensive troubleshooting section is provided. We established and validated a protocol for triple-detection size-exclusion chromatography that enables the inexperienced user to perform and analyse measurements of well-behaved protein/detergent complexes. More experienced users are provided with an example of a more sophisticated analysis procedure allowing mass determination under challenging separation conditions.