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 25
Externally publishedYes

Fingerprint

Glycosylphosphatidylinositols
Electrophoresis
Purification
Gels
Proteins
Sodium Dodecyl Sulfate
Detergents
Nonidet P-40
Tetrahymena
Alkaline Phosphatase
Polyacrylamide Gel Electrophoresis
Buffers
Agglomeration

ASJC Scopus subject areas

  • Biochemistry
  • Biophysics
  • Molecular Biology

Cite this

Purification of glycosylphosphatidylinositol-anchored proteins by modified Triton X-114 partitioning and preparative gel electrophoresis. / Ko, Young-Gyu; Thompson, Guy A.

In: Analytical Biochemistry, Vol. 224, No. 1, 25.01.1995, p. 166-172.

Research output: Contribution to journalArticle

@article{eb5fd518811e4eecba5a5ec51d3562f5,
title = "Purification of glycosylphosphatidylinositol-anchored proteins by modified Triton X-114 partitioning and preparative gel electrophoresis",
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.",
author = "Young-Gyu Ko and Thompson, {Guy A.}",
year = "1995",
month = "1",
day = "25",
doi = "10.1006/abio.1995.1024",
language = "English",
volume = "224",
pages = "166--172",
journal = "Analytical Biochemistry",
issn = "0003-2697",
publisher = "Academic Press Inc.",
number = "1",

}

TY - JOUR

T1 - Purification of glycosylphosphatidylinositol-anchored proteins by modified Triton X-114 partitioning and preparative gel electrophoresis

AU - Ko, Young-Gyu

AU - Thompson, Guy A.

PY - 1995/1/25

Y1 - 1995/1/25

N2 - 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.

AB - 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.

UR - http://www.scopus.com/inward/record.url?scp=0028869863&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0028869863&partnerID=8YFLogxK

U2 - 10.1006/abio.1995.1024

DO - 10.1006/abio.1995.1024

M3 - Article

C2 - 7710065

AN - SCOPUS:0028869863

VL - 224

SP - 166

EP - 172

JO - Analytical Biochemistry

JF - Analytical Biochemistry

SN - 0003-2697

IS - 1

ER -