On-Line Concentration of a Protein Using Denaturation by Sodium Dodecyl Sulfate

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  • JING Peng
    Department of Applied Chemistry, Graduate School of Engineering, Kyushu University
  • KANETA Takashi
    Department of Applied Chemistry, Graduate School of Engineering, Kyushu University
  • IMASAKA Totaro
    Department of Applied Chemistry, Graduate School of Engineering, Kyushu University

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抄録

A novel method for the on-column sample stacking of proteins is described. The strategy takes advantage of interactions between protein molecules and sodium dodecyl sulfate (SDS) monomers. A long plug of a protein sample (either acidic or basic) is injected into a capillary filled with a background electrolyte (BGE) containing SDS. When a potential is applied, the proteins interact with SDS monomers in the BGE to form protein-SDS complexes that migrate more slowly than the corresponding uncomplexed protein, resulting in protein stacking. Both acidic and basic proteins migrate at an almost identical electrophoretic velocity after stacking, which indicates that the protein-SDS complexes formed in the BGE zone have a similar charge/mass ratio. The mechanism of stacking was investigated using a sample consisting of a basic protein, lysozyme, and a small molecule, methylene blue. The findings clearly show that two interactions with SDS occur, a stepwise binding interaction between protein molecules and SDS monomers and an interaction in which the small molecules enter into micelles formed by SDS molecules. The method was also applied to the detection of a protein labeled with a fluorescent labeling reagent at trace levels. The labeled protein was detected even under labeling conditions where the labeling efficiency was too low to detect by short-plug injection.

収録刊行物

  • Analytical Sciences

    Analytical Sciences 21 (1), 37-42, 2005

    社団法人 日本分析化学会

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