Refractive index-based determination of detergent concentration and its application to the study of membrane proteins.
The concentration of detergent in membrane protein preparations can have a critical role on protein stability, function, and the potential for crystallization. Unfortunately, dialysis or protein concentration can lead to an unknown amount of detergent in the final membrane protein preparations. Here we present a method for the determination of detergent concentration based on refractive index of the detergent solution. This method was applied to quantitate the amount of detergent remaining in solution after concentration in various concentrators. We found that the ability of the tested detergents to pass through the molecular weight cutoff membrane correlates well with detergent micelle size. Therefore, the micelle size can be used as a rough guide to estimate the retention of a given detergent in various molecular weight cutoff concentrators. The refractive index method is exceptionally informative when coupled with size exclusion chromatography and light scattering, and can be used to determine the oligomeric state of the membrane protein, the size of a protein-associated micelle, as well as the amount and size of the unbound detergent micelle.
Strop
Pavel
P
Howard Hughes Medical Institute, Stanford, California 94305, USA.
Brunger
Axel T
AT
D002850:Chromatography, Gel; D003902:Detergents / Q000032:analysis* / Q000302:isolation & purification; D008565:Membrane Proteins / Q000737:chemistry* / Q000302:isolation & purification*; D008823:Micelles; D012015:Reference Standards; D012031:Refractometry / Q000379:methods* / Q000592:standards
D003902:Detergents; D008565:Membrane Proteins; D008823:Micelles