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<title>Enzymatic fluorescent labeling of 6-gala (neogala) series glycosphingolipids-oligosaccharides - Glycoscience Protocols (GlycoPODv2) - NCBI Bookshelf</title>
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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="Enzymatic fluorescent labeling of 6-gala (neogala) series glycosphingolipids-oligosaccharides" /><meta name="citation_publisher" content="Japan Consortium for Glycobiology and Glycotechnology" /><meta name="citation_date" content="2022/03/21" /><meta name="citation_author" content="Yohei Ishibashi" /><meta name="citation_author" content="Makoto Ito" /><meta name="citation_pmid" content="37590690" /><meta name="citation_fulltext_html_url" content="https://www.ncbi.nlm.nih.gov/books/NBK593955/" /><meta name="citation_keywords" content="fluorescent labeling" /><meta name="citation_keywords" content="endogalactosylceramidase (EGALC" /><meta name="citation_keywords" content="EGCase III)" /><meta name="citation_keywords" content="digalactosyldiacylglycerol (DGDG)" /><meta name="citation_keywords" content="transglycosylation" /><meta name="citation_keywords" content="6-gala (neogala) series GSLs" /><link rel="schema.DC" href="http://purl.org/DC/elements/1.0/" /><meta name="DC.Title" content="Enzymatic fluorescent labeling of 6-gala (neogala) series glycosphingolipids-oligosaccharides" /><meta name="DC.Type" content="Text" /><meta name="DC.Publisher" content="Japan Consortium for Glycobiology and Glycotechnology" /><meta name="DC.Contributor" content="Yohei Ishibashi" /><meta name="DC.Contributor" content="Makoto Ito" /><meta name="DC.Date" content="2022/03/21" /><meta name="DC.Identifier" content="https://www.ncbi.nlm.nih.gov/books/NBK593955/" /><meta name="description" content="High-performance liquid chromatography (HPLC), thin-layer chromatography (TLC), and capillary electrophoresis have been used for the separation and detection of oligosaccharides released from glycosphingolipids (GSLs). Since intact oligosaccharides exhibit no effective absorption in the UV-Vis region, chemical derivatization of oligosaccharides with 2-aminopyridine (AP), 2-aminobenzamide (AB), 2-aminobenzoic acid (AA), or 4-aminobenzoic ethyl ester (4-ABEE) is frequently used to confer fluorescence or UV-absorption to oligosaccharides for detection (1). However, chemical derivatization is somewhat time-consuming and requires purification that may lead to a loss of samples. In this section, the enzymatic derivatization of 6-gala (neogala) series glycolipid-derived oligosaccharides using a specific enzyme, endogalactosylceramidase (EGALC, EGCase III), is described." /><meta name="og:title" content="Enzymatic fluorescent labeling of 6-gala (neogala) series glycosphingolipids-oligosaccharides" /><meta name="og:type" content="book" /><meta name="og:description" content="High-performance liquid chromatography (HPLC), thin-layer chromatography (TLC), and capillary electrophoresis have been used for the separation and detection of oligosaccharides released from glycosphingolipids (GSLs). Since intact oligosaccharides exhibit no effective absorption in the UV-Vis region, chemical derivatization of oligosaccharides with 2-aminopyridine (AP), 2-aminobenzamide (AB), 2-aminobenzoic acid (AA), or 4-aminobenzoic ethyl ester (4-ABEE) is frequently used to confer fluorescence or UV-absorption to oligosaccharides for detection (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="_NBK593955_"><span class="title" itemprop="name">Enzymatic fluorescent labeling of 6-gala (neogala) series glycosphingolipids-oligosaccharides</span></h1><div class="contrib half_rhythm"><span itemprop="author">Yohei Ishibashi</span>, Ph.D.<div class="affiliation small">Kyushu Univ<div><span class="email-label">Email: </span><a href="mailto:dev@null" data-email="pj.ca.u-uhsuyk.rga@oyihsabihsi" class="oemail">pj.ca.u-uhsuyk.rga@oyihsabihsi</a></div></div><div class="small">Corresponding author.</div></div><div class="contrib half_rhythm"><span itemprop="author">Makoto Ito</span>, Ph.D.<div class="affiliation small">Kyushu Univ<div><span class="email-label">Email: </span><a href="mailto:dev@null" data-email="pj.ca.u-uhsuyk.rga@iotokam" class="oemail">pj.ca.u-uhsuyk.rga@iotokam</a></div></div><div class="small">Corresponding author.</div></div><p class="small">Created: <span itemprop="datePublished">October 5, 2021</span>; Last Revision: <span itemprop="dateModified">March 21, 2022</span>.</p></div><div class="body-content whole_rhythm" itemprop="text"><div id="g192-fluorolabel.Introduction"><h2 id="_g192-fluorolabel_Introduction_">Introduction</h2><p>High-performance liquid chromatography (HPLC), thin-layer chromatography (TLC), and capillary electrophoresis have been used for the separation and detection of oligosaccharides released from glycosphingolipids (GSLs). Since intact oligosaccharides exhibit no effective absorption in the UV-Vis region, chemical derivatization of oligosaccharides with 2-aminopyridine (AP), 2-aminobenzamide (AB), 2-aminobenzoic acid (AA), or 4-aminobenzoic ethyl ester (4-ABEE) is frequently used to confer fluorescence or UV-absorption to oligosaccharides for detection (<a class="bk_pop" href="#g192-fluorolabel.REF.1">1</a>). However, chemical derivatization is somewhat time-consuming and requires purification that may lead to a loss of samples. In this section, the enzymatic derivatization of 6-gala (neogala) series glycolipid-derived oligosaccharides using a specific enzyme, endogalactosylceramidase (EGALC, EGCase III), is described.</p><p>6-Gala series GSLs possessing R-Gal (α/β)1-6Galβ1-1’Cer have been found in some mollusks, pathogenic parasites, and fungi, but their physiological functions and metabolic pathways are not fully understood. In this section, we describe a new method to detect 6-gala series GSLs utilizing the specificity of a novel enzyme, EGALC, which is capable of hydrolyzing 6-gala series GSLs to produce intact oligosaccharides and ceramides (<a class="bk_pop" href="#g192-fluorolabel.REF.2">2</a>). EGALC catalyzes not only hydrolysis but also the transglycosylation reaction (<a class="bk_pop" href="#g192-fluorolabel.REF.3">3</a>). In the latter reaction, EGALC transfers oligosaccharides from the GSLs to acceptors, such as fluorescent 1-alkanols. Utilizing the transglycosylation reaction of EGALC, a specific, easy, fast, sensitive, and reproducible method for detecting 6-gala series GSLs was developed using NBD-pentanol as an acceptor (<a class="figpopup" href="/books/NBK593955/figure/g192-fluorolabel.F1/?report=objectonly" target="object" rid-figpopup="figg192fluorolabelF1" rid-ob="figobg192fluorolabelF1">Figure 1</a>) (<a class="bk_pop" href="#g192-fluorolabel.REF.4">4</a>). The fluorescent products (NBD-pentanol-conjugated 6-gala oligosaccharides) were separated and detected using TLC or HPLC with a fluorescent detector. Moreover, not only GSLs but also glycoglycerolipids having the R-Gal(α/β)1-6Galβ1-1’diacylglycerol structure, such as digalactosyldiacylglycerol (DGDG), can be detected using this method. This method was successfully applied to the detection of 6-gala series GSLs in a fungus (<i>Rhizopus oryzae</i>) and parasite (<i>Taenia crassiceps</i>) (<a class="bk_pop" href="#g192-fluorolabel.REF.4">4</a>). The method would be useful for the study of glycolipids, which share the R-Gal (α/β)1-6Gal structure. Novel glycolipid having ether-linked phytol and Galα1-6Gal moiety was discovered in green algae using this method (<a class="bk_pop" href="#g192-fluorolabel.REF.5">5</a>).</p></div><div id="g192-fluorolabel.Protocol"><h2 id="_g192-fluorolabel_Protocol_">Protocol</h2><p>This chapter describes the direct fluorescent labeling of 6-gala series GSL-oligosaccharides using transglycosylation reaction of EGALC, which hydrolyzes 6-gala series GSLs into intact oligosaccharides and ceramides. First, prepare fluorescent alkanols (NBD-pentanol), then incubate 6-gala series GSLs with EGALC in the presence of fluorescent alkanols, and finally analyze the fluorescent GSL-oligosaccharides using TLC or HPLC.</p><div id="g192-fluorolabel.Materials"><h3>Materials</h3><dl class="temp-labeled-list"><dt>1.</dt><dd><p class="no_top_margin">Total lipid fraction prepared using the method described in “Extraction of glycolipids”</p></dd><dt>2.</dt><dd><p class="no_top_margin">Pre-coated Silica gel 60 TLC plates (Merck Millipore, Billerica, MA, USA)</p></dd><dt>3.</dt><dd><p class="no_top_margin">5-Amino-1-pentanol (TCI, Tokyo, Japan)</p></dd><dt>4.</dt><dd><p class="no_top_margin">4-Fluoro-7nitro-2,1,3-benzoxadiazole (NBD-F) (Sigma-Aldrich, MO, USA)</p></dd><dt>5.</dt><dd><p class="no_top_margin">Sodium acetate buffer, pH 5.5</p></dd><dt>6.</dt><dd><p class="no_top_margin">Triton X-100 (Sigma-Aldrich)</p></dd><dt>7.</dt><dd><p class="no_top_margin">Chloroform (Nacalai Tesque Inc., Kyoto, Japan)</p></dd><dt>8.</dt><dd><p class="no_top_margin">Methanol (Nacalai Tesque, Inc.)</p></dd><dt>9.</dt><dd><p class="no_top_margin">Acetonitrile HPLC grade (Kanto Chemical Co. Inc., Tokyo, Japan)</p></dd><dt>10.</dt><dd><p class="no_top_margin">EGALC (<b>Note 1</b>)</p></dd><dt>11.</dt><dd><p class="no_top_margin">Ninhydrin reagent (Sigma-Aldrich)</p></dd></dl></div><div id="g192-fluorolabel.Instruments"><h3>Instruments</h3><dl class="temp-labeled-list"><dt>1.</dt><dd><p class="no_top_margin">Sep-Pak silica cartridge (Waters, MA, USA)</p></dd><dt>2.</dt><dd><p class="no_top_margin">Sep-Pak C18 cartridge (Waters, if necessary)</p></dd><dt>3.</dt><dd><p class="no_top_margin">HPLC with fluorescence detector</p></dd><dt>4.</dt><dd><p class="no_top_margin">Asahipak NH2P 50 4E, 4.6 × 250 mm (Shodex, Tokyo, Japan)</p></dd><dt>5.</dt><dd><p class="no_top_margin">AE-6935B Visirays (ATTO, Tokyo, Japan)</p></dd><dt>6.</dt><dd><p class="no_top_margin">Shimadzu CS-9300 TLC chromatoscanner with the fluorescence detector (SHIMADZU, Kyoto, Japan)</p></dd><dt>7.</dt><dd><p class="no_top_margin">Block heater</p></dd><dt>8.</dt><dd><p class="no_top_margin">Speed vac concentrator</p></dd><dt>9.</dt><dd><p class="no_top_margin">TLC developing chamber</p></dd></dl></div><div id="g192-fluorolabel.Methods"><h3>Methods</h3><dl class="temp-labeled-list"><dt>1.</dt><dd><p class="no_top_margin">Preparation of NBD-pentanol</p><dl class="temp-labeled-list"><dt>a.</dt><dd><p class="no_top_margin">Mix 25 μmol of NBD-F and 25 μmol of 5-amino-1-pentanol and incubate at 60°C for 1 min.</p></dd><dt>b.</dt><dd><p class="no_top_margin">Dry the mixture with a speed vac concentrator, dissolve the residue in 2 mL of chloroform, and apply the solution to a Sep-Pak silica cartridge equilibrated with chloroform.</p></dd><dt>c.</dt><dd><p class="no_top_margin">Elute NBD-pentanol from the cartridge with 5 mL of chloroform/methanol (9/1, v/v). Dry the eluent with speed vac concentrator and dissolve it in 2 mL of ethanol.</p></dd><dt>d.</dt><dd><p class="no_top_margin">Check the quality and quantity of purified NBD-pentanol by TLC with AE-6935B Visirays and with ninhydrin reagent to detect unreacted 5-amino-1-pentanol (<b>Note 2</b>).</p></dd></dl></dd><dt>2.</dt><dd><p class="no_top_margin">Transglycosylation reaction</p><dl class="temp-labeled-list"><dt>a.</dt><dd><p class="no_top_margin">Evaporate an appropriate amount of total lipids (>400 ng) and 20 nmol of NBD-pentanol in a 1.5-mL tube with a speed vac concentrator.</p></dd><dt>b.</dt><dd><p class="no_top_margin">Prepare 10 μL of a reaction mixture containing 10 μU of EGALC and 0.1% Triton X-100 in 50 mM of sodium acetate buffer, pH 5.5, and incubate the mixture at 37°C for 2 h.</p></dd><dt>c.</dt><dd><p class="no_top_margin">Add 80 μL of chloroform/methanol (2/1, v/v) and 10 μL of water. After vortexing for a few seconds, centrifuge the mixture at max speed for 3 min.</p></dd><dt>d.</dt><dd><p class="no_top_margin">Collect the upper phase containing NBD-pentanol-conjugated oligosaccharides, add 100 μL of chloroform/methanol/water (86/14/1, v/v/v), and centrifuge the mixture at max speed for 3 min (<b>Note 3</b>).</p></dd><dt>e.</dt><dd><p class="no_top_margin">Collect the second upper phase, combine the first and second upper phases, and dry it with speed vac concentrator. You can choose TLC or HPLC to detect the transglycosylation products (<b>Note 4</b>).</p></dd></dl></dd><dt>3.</dt><dd><p class="no_top_margin">TLC analysis of transglycosylation products</p><dl class="temp-labeled-list"><dt>a.</dt><dd><p class="no_top_margin">Dissolve the dried sample of Step 2e in 10 μL of methanol and apply it to a TLC plate.</p></dd><dt>b.</dt><dd><p class="no_top_margin">Develop the TLC plate with chloroform/methanol/0.02% CaCl<sub>2</sub> (5/4/1, v/v/v) for 30 min.</p></dd><dt>c.</dt><dd><p class="no_top_margin">Visualize transglycosylation products by AE-6935B Visirays or UV transilluminator.</p></dd><dt>d.</dt><dd><p class="no_top_margin">Quantify products with a Shimadzu CS-9300 TLC chromatoscanner with the fluorescence detector (excitation 475 nm).</p></dd></dl></dd><dt>4.</dt><dd><p class="no_top_margin">HPLC analysis of transglycosylation products</p><dl class="temp-labeled-list"><dt>a.</dt><dd><p class="no_top_margin">Set excitation and emission wavelengths of fluorescence detector to 470 and 530 nm, respectively.</p></dd><dt>b.</dt><dd><p class="no_top_margin">Dissolve the dried sample of Step 2e in 120 μL of acetonitrile/water (9/1, v/v) and centrifuge it at max speed for 5 min.</p></dd><dt>c.</dt><dd><p class="no_top_margin">Apply 100 μL of the supernatant to HPLC with Asahipak NH2P 50 4E column equilibrated with solvent A (acetonitrile/water [9/1, v/v]). HPLC gradient conditions are 0% solvent B (acetonitrile/water [1/1, v/v]) to 100% B in 25 min at a flow rate of 1.0 mL/min.</p></dd><dt>d.</dt><dd><p class="no_top_margin">Before performing the next run, equilibrate the column with 100% solvent A for 10 min.</p></dd></dl></dd></dl></div><div id="g192-fluorolabel.Notes"><h3>Notes</h3><dl class="temp-labeled-list"><dt>1.</dt><dd><p class="no_top_margin">EGALC is not commercially available presently. For requesting EGALC, please contact the author.</p></dd><dt>2.</dt><dd><p class="no_top_margin">If the purity of NBD-pentanol is not enough, further purification step using a C18 Sep-Pak cartridge is required.</p><dl class="temp-labeled-list"><dt>a.</dt><dd><p class="no_top_margin">Dry the mixture with a speed vac concentrator, dissolve the residue in 2 mL of water/methanol (9/1, v/v), and apply the solution on a Sep-Pak C18 cartridge conditioned by methanol and equilibrated with water/methanol (9/1, v/v).</p></dd><dt>b.</dt><dd><p class="no_top_margin">Wash the cartridge with 5 mL of water/methanol (9/1, v/v) and water/methanol (5/1, v/v) then elute NBD-pentanol from the cartridge with 5 mL of methanol. Dry the eluent with a speed vac concentrator and dissolve it in 2 mL of ethanol.</p></dd></dl></dd><dt>3.</dt><dd><p class="no_top_margin">Removal of unreacted excess reagent is necessary for the appropriate separation and quantification of fluorescent derivatives. For this purpose, Folch’s partition can be used wherein NBD-pentanol is moved to the organic phase, while almost all fluorescent oligosaccharides are moved to the water phase without a drop in yield.</p></dd><dt>4.</dt><dd><p class="no_top_margin">The limit of detection (LOD) of fluorescent-GLSs by TLC is ~1.5 pmol (S/N = 3) and that of HPLC is 50 fmol (S/N = 5). The detection of the labeled oligosaccharide is linear from 4 pmol to 60 pmol in TLC and from 250 fmol to 60 pmol in HPLC.he limit of detection (LOD) of fluorescent-GLSs by TLC is ~1.5 pmol (S/N = 3) and that of HPLC is 50 fmol (S/N = 5). The detection of the labeled oligosaccharide is linear from 4 pmol to 60 pmol in TLC and from 250 fmol to 60 pmol in HPLC.</p></dd></dl></div></div><div id="g192-fluorolabel.References"><h2 id="_g192-fluorolabel_References_">References</h2><dl class="temp-labeled-list"><dt>1.</dt><dd><div class="bk_ref" id="g192-fluorolabel.REF.1">Pabst M, Kolarich D, Pöltl G, Dalik T, Lubec G, Hofinger A, Altmann F. Comparison of fluorescent labels for oligosaccharides and introduction of a new postlabeling purification method. <span><span class="ref-journal">Anal Biochem. </span>2009 Jan 15;<span class="ref-vol">384</span>(2):263–73.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/18940176" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 18940176</span></a>] [<a href="http://dx.crossref.org/10.1016/j.ab.2008.09.041" ref="pagearea=cite-ref&targetsite=external&targetcat=link&targettype=uri">CrossRef</a>]</div></dd><dt>2.</dt><dd><div class="bk_ref" id="g192-fluorolabel.REF.2">Ishibashi Y, Nakasone T, Kiyohara M, Horibata Y, Sakaguchi K, Hijikata A, Ichinose S, Omori A, Yasui Y, Imamura A, Ishida H, Kiso M, Okino N, Ito M. A novel endoglycoceramidase hydrolyzes oligogalactosylceramides to produce galactooligosaccharides and ceramides. <span><span class="ref-journal">J Biol Chem. </span>2007 Apr 13;<span class="ref-vol">282</span>(15):11386–96.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/17244618" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 17244618</span></a>] [<a href="http://dx.crossref.org/10.1074/jbc.M608445200" ref="pagearea=cite-ref&targetsite=external&targetcat=link&targettype=uri">CrossRef</a>]</div></dd><dt>3.</dt><dd><div class="bk_ref" id="g192-fluorolabel.REF.3">Ishibashi Y, Kiyohara M, Okino N, Ito M. Synthesis of fluorescent glycosphingolipids and neoglycoconjugates which contain 6-gala oligosaccharides using the transglycosylation reaction of a novel endoglycoceramidase (EGALC). <span><span class="ref-journal">J Biochem. </span>2007 Aug;<span class="ref-vol">142</span>(2):239–46.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/17567653" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 17567653</span></a>] [<a href="http://dx.crossref.org/10.1093/jb/mvm125" ref="pagearea=cite-ref&targetsite=external&targetcat=link&targettype=uri">CrossRef</a>]</div></dd><dt>4.</dt><dd><div class="bk_ref" id="g192-fluorolabel.REF.4">Ishibashi Y, Nagamatsu Y, Meyer S, Imamura A, Ishida H, Kiso M, Okino N, Geyer R, Ito M. Transglycosylation-based fluorescent labeling of 6-gala series glycolipids by endogalactosylceramidase. <span><span class="ref-journal">Glycobiology. </span>2009 Jul;<span class="ref-vol">19</span>(7):797–807.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/19389917" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 19389917</span></a>] [<a href="http://dx.crossref.org/10.1093/glycob/cwp051" ref="pagearea=cite-ref&targetsite=external&targetcat=link&targettype=uri">CrossRef</a>]</div></dd><dt>5.</dt><dd><div class="bk_ref" id="g192-fluorolabel.REF.5">Ishibashi Y, Nagamatsu Y, Miyamoto T, Matsunaga N, Okino N, Yamaguchi K, Ito M. A novel ether-linked phytol-containing digalactosylglycerolipid in the marine green alga, Ulva pertusa. <span><span class="ref-journal">Biochem Biophys Res Commun. </span>2014 Oct 3;<span class="ref-vol">452</span>(4):873–80.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/25157808" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 25157808</span></a>] [<a href="http://dx.crossref.org/10.1016/j.bbrc.2014.08.056" ref="pagearea=cite-ref&targetsite=external&targetcat=link&targettype=uri">CrossRef</a>]</div></dd></dl></div><h2 id="NBK593955_footnotes">Footnotes</h2><dl class="temp-labeled-list small"><dt></dt><dd><div id="g192-fluorolabel.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="g192-fluorolabel.F1" class="figure bk_fig"><div class="graphic"><a href="/core/lw/2.0/html/tileshop_pmc/tileshop_pmc_inline.html?title=Figure%201%3A%20.%20The%20scheme%20of%20transglycosylation-based%20fluorescent%20labeling%20of%206-gala%20series%20glycosphingolipids%20using%20endogalactosylceramidase.&p=BOOKS&id=593955_g192-fluorolabel-Image001.jpg" target="tileshopwindow" class="inline_block pmc_inline_block ts_canvas img_link" title="Click on image to zoom"><div class="ts_bar small" title="Click on image to zoom"></div><img src="/books/NBK593955/bin/g192-fluorolabel-Image001.jpg" alt="Figure 1: . The scheme of transglycosylation-based fluorescent labeling of 6-gala series glycosphingolipids using endogalactosylceramidase." class="tileshop" title="Click on image to zoom" /></a></div><h3><span class="label">Figure 1: </span></h3><div class="caption"><p>The scheme of transglycosylation-based fluorescent labeling of 6-gala series glycosphingolipids using endogalactosylceramidase.</p></div><div class="permissions">This figure was originally published in Glycobiology. 19(7):797–807. 2009 "Transglycosylation-based fluorescent labeling of 6-gala series glycolipids by endogalactosylceramidase” Ishibashi Y. et al. Oxford University Press.<p class="small">We obtained the license from Oxford University Press, License Number 5194051049780</p></div></div></div></div><div id="bk_toc_contnr"></div></div></div>
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