title: Characterising protein/detergent complexes by triple-detection size-exclusion chromatography.
abstract: BACKGROUND
  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.
  
  
  RESULTS
  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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Molecular Biophysics, University of Kaiserslautern, Erwin-Schrödinger-Str. 13, 67663 Kaiserslautern, Germany.
authors.forename: Katharina
authors.initials: K
authors.lastname: Gimpl
authors.affiliation: Molecular Biophysics, University of Kaiserslautern, Erwin-Schrödinger-Str. 13, 67663 Kaiserslautern, Germany.
authors.forename: Jessica
authors.initials: J
authors.lastname: Klement
authors.affiliation: Molecular Biophysics, University of Kaiserslautern, Erwin-Schrödinger-Str. 13, 67663 Kaiserslautern, Germany.
authors.forename: Sandro
authors.initials: S
authors.lastname: Keller
keywords: Absolute mass determination; Detergent micelles; Membrane proteins; Multiple detection; Static light scattering