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Refractive index-based determination of detergent concentration and its application to the study of membrane proteins

Authors

  • Pavel Strop,

    1. Howard Hughes Medical Institute and Departments of Molecular and Cellular Physiology, Neurology and Neurological Sciences, and Stanford Synchrotron Radiation Laboratory, Stanford University, James H. Clark Center, Stanford, California 94305, USA
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  • Axel T. Brunger

    Corresponding author
    1. Howard Hughes Medical Institute and Departments of Molecular and Cellular Physiology, Neurology and Neurological Sciences, and Stanford Synchrotron Radiation Laboratory, Stanford University, James H. Clark Center, Stanford, California 94305, USA
    • Stanford University School of Medicine/HHMI, James H. Clark Center E300-C, 318 Campus Dr., Stanford, CA 94305, USA; fax: (650) 736-1961.
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Abstract

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.

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