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<meta name="robots" content="INDEX,FOLLOW,NOARCHIVE" /><meta name="citation_inbook_title" content="Glycoscience Protocols (GlycoPODv2) [Internet]" /><meta name="citation_title" content="Partitioning of glycosylphosphatidylinositol (GPI)-anchored proteins with Triton X-114" /><meta name="citation_publisher" content="Japan Consortium for Glycobiology and Glycotechnology" /><meta name="citation_date" content="2022/03/25" /><meta name="citation_author" content="Morihisa Fujita" /><meta name="citation_author" content="Yusuke Maeda" /><meta name="citation_author" content="Taroh Kinoshita" /><meta name="citation_pmid" content="37590577" /><meta name="citation_fulltext_html_url" content="https://www.ncbi.nlm.nih.gov/books/NBK593825/" /><meta name="citation_keywords" content="glycosylphosphatidylinositol (GPI)" /><meta name="citation_keywords" content="GPI anchor" /><meta name="citation_keywords" content="GPI-anchored proteins" /><meta name="citation_keywords" content="phosphatidylinositol-specific phospholipase C (PI-PLC)" /><link rel="schema.DC" href="http://purl.org/DC/elements/1.0/" /><meta name="DC.Title" content="Partitioning of glycosylphosphatidylinositol (GPI)-anchored proteins with Triton X-114" /><meta name="DC.Type" content="Text" /><meta name="DC.Publisher" content="Japan Consortium for Glycobiology and Glycotechnology" /><meta name="DC.Contributor" content="Morihisa Fujita" /><meta name="DC.Contributor" content="Yusuke Maeda" /><meta name="DC.Contributor" content="Taroh Kinoshita" /><meta name="DC.Date" content="2022/03/25" /><meta name="DC.Identifier" content="https://www.ncbi.nlm.nih.gov/books/NBK593825/" /><meta name="description" content="Many eukaryotic cell surface proteins with various functions are anchored to the membrane via glycosylphosphatidylinositol (GPI). 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Once GPI moieties are cleaved by enzymes, including phosphatidylinositol-specific phospholipase C (PI-PLC) and GPI-specific phospholipase D (GPI-PLD), the protein parts no longer partition into the detergent phase and recover into the aqueous phase (1, 2). This characteristic of partitioning behavior is useful when the GPI anchor is present (Note 1). 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<div class="pre-content"><div><div class="bk_prnt"><p class="small">NCBI Bookshelf. A service of the National Library of Medicine, National Institutes of Health.</p><p>Nishihara S, Angata K, Aoki-Kinoshita KF, et al., editors. Glycoscience Protocols (GlycoPODv2) [Internet]. Saitama (JP): Japan Consortium for Glycobiology and Glycotechnology; 2021-. </p></div></div></div>
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<div class="main-content lit-style" itemscope="itemscope" itemtype="http://schema.org/CreativeWork"><div class="meta-content fm-sec"><h1 id="_NBK593825_"><span class="title" itemprop="name">Partitioning of glycosylphosphatidylinositol (GPI)-anchored proteins with Triton X-114</span></h1><div class="contrib half_rhythm"><span itemprop="author">Morihisa Fujita</span>, Ph.D.<div class="affiliation small">Institute for Glyco-core Research, Gifu University<div><span class="email-label">Email: </span><a href="mailto:dev@null" data-email="pj.ca.u-ufig@atijufm" class="oemail">pj.ca.u-ufig@atijufm</a></div></div><div class="small">Corresponding author.</div></div><div class="contrib half_rhythm"><span itemprop="author">Yusuke Maeda</span>, M.D./Ph.D.<div class="affiliation small">Research Institute for Microbial Diseases, Osaka University<div><span class="email-label">Email: </span><a href="mailto:dev@null" data-email="pj.ca.u-akaso.nekib@adeamy" class="oemail">pj.ca.u-akaso.nekib@adeamy</a></div></div></div><div class="contrib half_rhythm"><span itemprop="author">Taroh Kinoshita</span>, Ph.D.<div class="affiliation small">Research Institute for Microbial Diseases, Osaka University<div><span class="email-label">Email: </span><a href="mailto:dev@null" data-email="pj.ca.u-akaso.nekib@ihsonikt" class="oemail">pj.ca.u-akaso.nekib@ihsonikt</a></div></div></div><p class="small">Created: <span itemprop="datePublished">September 17, 2021</span>; Last Revision: <span itemprop="dateModified">March 25, 2022</span>.</p></div><div class="body-content whole_rhythm" itemprop="text"><div id="g180-partgpitritonx.Introduction"><h2 id="_g180-partgpitritonx_Introduction_">Introduction</h2><p>Many eukaryotic cell surface proteins with various functions are anchored to the membrane via glycosylphosphatidylinositol (GPI). Triton X-114 is a nonionic detergent with a low cloud point (23°C); an indicator for phase separation of hydrophilic proteins from amphiphilic and hydrophobic proteins. At temperatures above the cloud point, detergent solutions separate into aqueous and detergent-enriched phases. GPI-anchored proteins are usually separated into detergent phases. Once GPI moieties are cleaved by enzymes, including phosphatidylinositol-specific phospholipase C (PI-PLC) and GPI-specific phospholipase D (GPI-PLD), the protein parts no longer partition into the detergent phase and recover into the aqueous phase (<a class="bk_pop" href="#g180-partgpitritonx.REF.1">1</a>, <a class="bk_pop" href="#g180-partgpitritonx.REF.2">2</a>). This characteristic of partitioning behavior is useful when the GPI anchor is present (<b>Note 1</b>). Here, we describe a protocol for the partitioning of GPI-anchored proteins with Triton X-114 combined with PI-PLC treatment.</p></div><div id="g180-partgpitritonx.Protocol"><h2 id="_g180-partgpitritonx_Protocol_">Protocol</h2><p>In the following protocol, we first described the preparation of 12% Triton X-114 solution (Method 1) and then explained the partitioning of GPI-anchored proteins using Triton X-114 (Method 2).</p><div id="g180-partgpitritonx.Materials"><h3>Materials</h3><dl class="temp-labeled-list"><dt>1.</dt><dd><p class="no_top_margin">Triton X-114</p></dd><dt>2.</dt><dd><p class="no_top_margin">Phosphate-buffered saline (PBS)</p></dd><dt>3.</dt><dd><p class="no_top_margin">Acetone</p></dd><dt>4.</dt><dd><p class="no_top_margin">PI-PLC (100 units/mL, P6466, Thermo Fisher Scientific, Waltham, MA)</p></dd><dt>5.</dt><dd><p class="no_top_margin">50% glycerol</p></dd><dt>6.</dt><dd><p class="no_top_margin">Suspension buffer (20 mM Tris-HCl (pH 7.4), 150 mM NaCl, 5 mM EDTA, and protease inhibitor cocktail)</p></dd><dt>7.</dt><dd><p class="no_top_margin">PI-PLC reaction buffer (20 mM Tris-HCl (pH 7.4), 0.1% Triton X-100, and protease inhibitor cocktail)</p></dd></dl></div><div id="g180-partgpitritonx.Instruments"><h3>Instruments</h3><dl class="temp-labeled-list"><dt>1.</dt><dd><p class="no_top_margin">Incubator (37°C)</p></dd><dt>2.</dt><dd><p class="no_top_margin">Centrifuge</p></dd></dl></div><div id="g180-partgpitritonx.Methods"><h3>Methods</h3><dl class="temp-labeled-list"><dt>1.</dt><dd><p class="no_top_margin">Preparation of 12% Triton X-114 solution</p><dl class="temp-labeled-list"><dt>a.</dt><dd><p class="no_top_margin">Dissolve 1.5 g of Triton X-114 in 50 mL of PBS on ice (or in a cold room).</p></dd><dt>b.</dt><dd><p class="no_top_margin">Warm the solution at 37°C until the solution becomes turbid.</p></dd><dt>c.</dt><dd><p class="no_top_margin">Centrifuge at 1,000 ×<i>g</i> for 10 min at room temperature.</p></dd><dt>d.</dt><dd><p class="no_top_margin">Remove and discard the upper phase (aqueous phase).</p></dd><dt>e.</dt><dd><p class="no_top_margin">Redissolve the lower phase (detergent phase) in an equal volume of cold-PBS and mix on ice.</p></dd><dt>f.</dt><dd><p class="no_top_margin">Repeat Steps 1b to 1e three times (The final detergent phase contains around 12% Triton X-114.).</p></dd><dt>g.</dt><dd><p class="no_top_margin">Store at 4°C until further use.</p></dd></dl></dd><dt>2.</dt><dd><p class="no_top_margin">Partitioning of GPI-anchored proteins with Triton X-114</p><dl class="temp-labeled-list"><dt>a.</dt><dd><p class="no_top_margin">Suspend cells (2 × 10<sup>6</sup>) in 1 mL of PBS.</p></dd><dt>b.</dt><dd><p class="no_top_margin">Centrifuge at 300 ×<i>g</i> for 3 min and resuspend the pellet in 100 µL of the suspension buffer.</p></dd><dt>c.</dt><dd><p class="no_top_margin">Add 20 µL of 12% Triton X-114 solution (2% final concentration), vortex well, and incubate on ice for 15 min.</p></dd><dt>d.</dt><dd><p class="no_top_margin">Centrifuge at 13,000 ×<i>g</i> for 15 min at 4°C to remove cell debris and transfer the supernatant to a new tube.</p></dd><dt>e.</dt><dd><p class="no_top_margin">Add 8-fold volume of ice-cold acetone and incubate for 1 h at −80°C.</p></dd><dt>f.</dt><dd><p class="no_top_margin">Centrifuge at 13,000 ×<i>g</i> for 15 min at 4°C, discard the supernatant, and dry up the protein pellet.</p></dd><dt>g.</dt><dd><p class="no_top_margin">Resuspend protein pellet in 200 µL of PI-PLC reaction buffer.</p></dd><dt>h.</dt><dd><p class="no_top_margin">Divide the suspension into two (100 µL each) and add 1 µL of PI-PLC (final: 1 unit/mL) or 50% glycerol (control).</p></dd><dt>i.</dt><dd><p class="no_top_margin">Incubate at 37°C for 1 h.</p></dd><dt>j.</dt><dd><p class="no_top_margin">Add 20 µL of 12% Triton X-114 solution (2% final concentration), vortex well, and incubate on ice for 10 min.</p></dd><dt>k.</dt><dd><p class="no_top_margin">Centrifuge at 1,000 ×<i>g</i> for 10 min at 37°C (alternatively, use a compact centrifuge, such as Chibitan [Merck, Darmstadt, Germany, 5,600 ×<i>g</i> for 7 min], in the incubator at 37°C).</p></dd><dt>l.</dt><dd><p class="no_top_margin">Transfer the upper phase to a new tube (upper phase: aqueous phase and lower phase: detergent phase).</p></dd><dt>m.</dt><dd><p class="no_top_margin">Add 100 µL of warmed PI-PLC reaction buffer to the lower phase, followed by 10 s of vigorous vortex. Incubate at 37°C for 2 min and remove the upper phase using centrifugation at 1,000 ×<i>g</i> for 10 min at 37°C (wash detergent phase to remove the contaminants from aqueous phase).</p></dd><dt>n.</dt><dd><p class="no_top_margin">Add 20 µL of Triton X-114 to aqueous phase (Step l), add 100 µL of PI-PLC reaction buffer to detergent phase (Step m) (to keep sample conditions and volumes consistent).</p></dd><dt>o.</dt><dd><p class="no_top_margin">Analyze by western blotting (<a class="figpopup" href="/books/NBK593825/figure/g180-partgpitritonx.F1/?report=objectonly" target="object" rid-figpopup="figg180partgpitritonxF1" rid-ob="figobg180partgpitritonxF1">Figure 1</a>). If necessary, immunoprecipitate proteins of interest before western blotting.</p></dd></dl></dd></dl></div><div id="g180-partgpitritonx.Note"><h3>Note</h3><dl class="temp-labeled-list"><dt>1.</dt><dd><p class="no_top_margin">Inositol-acylated GPI anchors are not cleaved by PI-PLC. During GPI biosynthesis in the endoplasmic reticulum (ER), an acyl chain is intermediately transferred to the 2-position of inositol on GPI. Therefore, GPI intermediates are resistant to PI-PLC. Soon after GPI attachment to proteins, the acyl chain is usually eliminated by GPI-inositol deacylase PGAP1 in the ER. The deacylated GPI anchors then become sensitive to PI-PLC. Therefore, many GPI-anchored proteins on mammalian cells are usually sensitive to PI-PLC. However, GPI-anchored proteins on human and mouse erythrocytes are resistant to PI-PLC treatment because the GPI structures maintain the acyl chain on the 2-position of the inositol.</p></dd></dl></div></div><div id="g180-partgpitritonx.References"><h2 id="_g180-partgpitritonx_References_">References</h2><dl class="temp-labeled-list"><dt>1.</dt><dd><div class="bk_ref" id="g180-partgpitritonx.REF.1">Doering TL, Englund PT, Hart GW. Detection of glycophospholipid anchors on proteins. Curr Protoc Mol Biol. 2001;Chapter 17:Unit17 8. doi: 10.1002/0471142727.mb1708s22. PMID: 18265164. [<a href="https://pubmed.ncbi.nlm.nih.gov/18265164" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 18265164</span></a>] [<a href="http://dx.crossref.org/10.1002/0471142727.mb1708s22" ref="pagearea=cite-ref&targetsite=external&targetcat=link&targettype=uri">CrossRef</a>]</div></dd><dt>2.</dt><dd><div class="bk_ref" id="g180-partgpitritonx.REF.2">Tanaka S, Maeda Y, Tashima Y, Kinoshita T. Inositol deacylation of glycosylphosphatidylinositol-anchored proteins is mediated by mammalian PGAP1 and yeast Bst1p. <span><span class="ref-journal">J Biol Chem. </span>2004;<span class="ref-vol">279</span>(14):14256–63.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/14734546" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 14734546</span></a>] [<a href="http://dx.crossref.org/10.1074/jbc.M313755200" ref="pagearea=cite-ref&targetsite=external&targetcat=link&targettype=uri">CrossRef</a>]</div></dd></dl></div><h2 id="NBK593825_footnotes">Footnotes</h2><dl class="temp-labeled-list small"><dt></dt><dd><div id="g180-partgpitritonx.FN1"><p class="no_top_margin">The authors declare no competing or financial interests.</p></div></dd></dl><div class="bk_prnt_sctn"><h2>Figures</h2><div class="whole_rhythm bk_prnt_obj bk_first_prnt_obj"><div id="g180-partgpitritonx.F1" class="figure bk_fig"><div class="graphic"><img src="/books/NBK593825/bin/g180-partgpitritonx-Image001.jpg" alt="Figure 1: . Phosphatidylinositol-specific phospholipase C (PI-PLC) sensitivity of extracted FLAG-tagged CD59." /></div><h3><span class="label">Figure 1: </span></h3><div class="caption"><p>Phosphatidylinositol-specific phospholipase C (PI-PLC) sensitivity of extracted FLAG-tagged CD59.</p><p>Chinese hamster ovary (CHO)-K1 cells were transiently transfected with FLAG-tagged CD59 cDNAs, treated with (+) or without (−) PI-PLC, lysed in 2% Triton X-114 on ice, and partitioned into the aqueous (A) and detergent (D) phases. FLAG-tagged CD59 proteins were immunoprecipitated from the detergent and aqueous phases with anti-FLAG beads and analyzed by sodium dodecyl–sulfate polyacrylamide gel electrophoresis/western blotting under reducing conditions using an anti-FLAG antibody (<a class="bk_pop" href="#g180-partgpitritonx.REF.2">2</a>).</p></div></div></div></div><div id="bk_toc_contnr"></div></div></div>
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