Characterising protein/detergent complexes by triple-detection size-exclusion chromatography

Biological Procedures Online

Methodology

Sandro Keller¹  Jessica Klement¹  Katharina Gimpl¹ 

[1] Molecular Biophysics, University of Kaiserslautern, Erwin-Schrödinger-Str. 13, 67663, Kaiserslautern, Germany;

关键词: Membrane proteins;    Detergent micelles;    Absolute mass determination;    Multiple detection;    Static light scattering;   

DOI  :  10.1186/s12575-015-0031-9

 received in 2015-10-29, accepted in 2015-12-29,  发布年份 2016

来源: Springer

【 摘 要 】

BackgroundIn 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.ResultsWe 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.ConclusionsWe 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.

【 授权许可】

CC BY   
© Gimpl et al. 2016