Purification of Glycosylphosphatidylinositol-Anchored Proteins by Modified Triton X-114 Partitioning and Preparative Gel Electrophoresis

Young Gyu Ko, Guy A. Thompson

Research output: Contribution to journalArticle

34 Citations (Scopus)

Abstract

Triton X-114 phase partitioning has frequently been used to obtain preparations enriched in glycosylphosphatidylinositol (GPI)-anchored proteins and other hydrophobic proteins from crude cellular homogenates. We have developed a new modification of this phase-partitioning technique which allows two distinct GPI-anchored proteins of Tetrahymena mimbres to be separated from other hydrophobic as well as hydrophilic proteins and recovered in ∼90% yield. The unique feature of the new method is a 24-h incubation of the first Triton X-114 extract at -20°C. This improves the partitioning of GPI-anchored proteins into the detergent phase while promoting the aggregation of other hydrophobic proteins. Individual GPI-anchored proteins in the detergent phase are then purified to near homogeneity by one-step preparative sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. By substituting Triton X-114/water mixtures for the conventional SDS buffer used to collect proteins eluting from the preparative gel, the purified GPI-anchored protein can be rapidly concentrated from relatively large volumes of eluate by phase partitioning at 32°C. The method is also effective in separating mammalian GPI-anchored alkaline phosphatase from other proteins. It is likely to be of general utility in characterizing the GPI anchor structures associated with nonabundant and abundant GPI-anchored proteins coexisting within the same cell type.

Original languageEnglish
Pages (from-to)166-172
Number of pages7
JournalAnalytical Biochemistry
Volume224
Issue number1
DOIs
Publication statusPublished - 1995 Jan
Externally publishedYes

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Molecular Biology
  • Cell Biology

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