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 language | English |
|---|---|
| Pages (from-to) | 166-172 |
| Number of pages | 7 |
| Journal | Analytical Biochemistry |
| Volume | 224 |
| Issue number | 1 |
| DOIs | |
| Publication status | Published - 1995 Jan 25 |
| Externally published | Yes |
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ASJC Scopus subject areas
- Biochemistry
- Biophysics
- Molecular Biology
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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 journal › Article
}
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.
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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 -