Isolation of an integral membrane glycoprotein by chloroform-methanol extraction and C3-reversed-phase high-performance liquid chromatography

Kurt R. Brunden, Carole T. Berg, Joseph F. Poduslo

Research output: Contribution to journalArticle

19 Scopus citations

Abstract

Methodology is presented for the isolation of integral membrane proteins and applied to the purification of the major myelin glycoprotein, P0. This isolation scheme depends on the detergent solubilization of an isoosmotically extracted membrane fraction from sciatic nerve endoneurium, followed by the removal of lipids and detergent by chloroform/methanol extraction. The resulting membrane proteins are readily dissolved in acetic acid/water ( 1 1) and directly analyzed by reversed-phase high-performance liquid chromatography. The hydrophobic nature of the intrinsic membrane protein mixture results in strong binding to a C8 stationary phase, leading to poor resolution and yields. These problems can be eliminated by employing a C3 alkylsilane column, thereby allowing separation of the protein components and the isolation of P0. The purified P0 has an amino-terminal sequence that matches that predicted from nucleotide sequencing, and the glycoprotein contains the expected amount of sialic acid. This latter finding indicates that the isolation procedure is not detrimental to the complex-type oligosaccharide structure of P0 and should make the methodology readily applicable to the purification of other integral membrane proteins and glycoproteins.

Original languageEnglish (US)
Pages (from-to)474-481
Number of pages8
JournalAnalytical Biochemistry
Volume164
Issue number2
DOIs
StatePublished - Aug 1 1987

Keywords

  • C reversed-phase high-performance liquid chromatography
  • P
  • chloroform/methanol extraction
  • integral membrane proteins
  • myelin glycoprotein
  • protein sequencing

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Molecular Biology
  • Cell Biology

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