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The Isolation of Detergent-Resistant Lipid Rafts for Two-Dimensional Electrophoresis

  • Ki-Bum Kim
  • Jae-Seon Lee
  • Young-Gyu Ko
Part of the Methods in Molecular Biology™ book series (MIMB, volume 424)

Summary

Because lipid rafts are plasma membrane platforms mediating various cellular events such as in signal transduction, immunological response, pathogen invasion, and neurodegenerative diseases, protein identification in the rafts could provide important information to study their function. Here, we present an optimized method to isolate detergent-resistant lipid rafts that are subsequently analyzed by two-dimensional electrophoresis (2-DE). Lipid rafts were isolated based on their two distinct biochemical properties such as Triton X-100 insolubility and low density. To solubilize completely the proteins embedded in lipid rafts, sample lysis buffer (9 M urea, 2 M thiourea, 100 mM DTT, 2% CHAPS (w/v), 60 mM n-octyl -D-glucopyranoside, 2% IPG buffer) was applied to the isolated rafts. This method was found to be the most suitable choice for obtaining 2-DE profile of lipid raft proteome from various cells and tissues. We expect that this method could provide the way to dissect the function of raft-associated proteins and to gain a comprehensive insight upon various cellular events mediated through lipid rafts, the specialized domains in cell surface.

Key Words

detergent-resistant lipid rafts Two-dimensional electrophoresis 

Notes

Acknowledgments

This work was supported by grants awarded to YK from KOSEF (R01-2004-000-10765-0), Korea Research Foundation (KRF-2006-311-C00407), and Top Brand Project of Korea Basic Science Institute.

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Copyright information

© Humana Press, a part of Springer Science+Business Media, LLC 2008

Authors and Affiliations

  • Ki-Bum Kim
    • 1
  • Jae-Seon Lee
    • 1
  • Young-Gyu Ko
    • 1
  1. 1.Korea UniversitySeoulKorea

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