Sialic acids were released from platelet GPIbα by treating the extract with acetic acid (2N final concentration) at 80°C for 3 h, filtered through a 10kD centrifugal filter (Microcon), and dried using a vacuum concentrator (SpeedVac). The released sialic acids were labeled with 1,2-diamino-4, 5-methylenedioxybenzene (DMB, Sigma Aldrich) for 2.5 h at 50°C [65 (link)]. HPLC analysis was performed using a Dionex UltiMate 3000 system with an Acclaim C18 column (ThermoFisher) under isocratic elution in 7% methanol, 7% acetonitrile, and 86% water. Sialic acid standards were derived from commercially available bovine submaxillary mucin, Neu5Gc and Neu5Ac (Sigma Aldrich) as well as from normal horse serum.
Neu5gc
Manufactured by Merck Group
Sourced in United States, Australia
Neu5Gc is a laboratory product manufactured by Merck Group. It is a sialic acid derivative that can be used in various research applications. The core function of Neu5Gc is to serve as a biochemical standard and analytical tool.
Automatically generated - may contain errors
Lab products found in correlation
Neu5ac, by Merck Group (5 mentions)
Acclaim c18 column, by Thermo Fisher Scientific (2 mentions)
1 phenyl 3 methyl 5 pyrazolone, by Merck Group (1 mentions)
Sephacryl s 300, by GE Healthcare (1 mentions)
Dulbecco s modified eagle medium dmem, by Thermo Fisher Scientific (1 mentions)
Ammonium formate, by Thermo Fisher Scientific (1 mentions)
Acetonitrile, by Thermo Fisher Scientific (1 mentions)
Trifluoroacetic acid tfa, by Thermo Fisher Scientific (1 mentions)
Accucore hilic column, by Thermo Fisher Scientific (1 mentions)
Methanol, by Merck Group (1 mentions)
Chloroform, by Merck Group (1 mentions)
Dionex ultimate 3000 system, by Thermo Fisher Scientific (1 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
Pre made phosphate buffer solution, by Merck Group (1 mentions)
Tryple, by Thermo Fisher Scientific (1 mentions)
Hplc grade ethanol, by Merck Group (1 mentions)
Hydrochloric acid (hcl), by Merck Group (1 mentions)
Sodium hydroxide (naoh), by Merck Group (1 mentions)
8 protocols using neu5gc
1
Analytical Quantification of Platelet Sialic Acids
2
Extraction and Characterization of Cod Sialic Acids
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Fresh mature female G. morhua were purchased from Oriental Ocean Company of China and stored at −80 °C until further use. QFF and Sephacryl S-300 were obtained from GE Healthcare (Fairfield, CT, USA). Neu5Ac, Neu5Gc, D2O, 1-phenyl-3-methyl-5-pyrazolone and dextran standards were purchased from Sigma (St. Louis, MO, USA). Dulbecco’s modified Eagle medium (DMEM) was obtained from Gibco (Gaithersburg, MD, USA). Fetal bovine serum was from HyClone (Logan, UT, USA).
3
HILIC-based Glycan Analysis
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The Accucore HILIC column (150 mm × 4.6 mm, 2.6 μm particles) was obtained from Thermo Scientific (Waltham, MA) and connected with an Accucore HILIC precolumn (10 mm × 4.6 mm, 2.6 μm). Ammonium formate, trifluoroacetic acid (TFA), and acetonitrile were obtained from Fisher Scientific (Carlbad, CA). Neu5Ac-D-1,2,3-13C3, KDN, Neu5Ac, Neu5Gc, and methanol were purchased from Sigma-Aldrich (St. Louis, MI), and chloroform was obtained from EMD Millipore (Burlington, MA). Milli-Q water (GenPure Pro ultrapure water system with UV-photo-oxidation and TOC monitor, by Thermo Scientific Inc) was used throughout the study protocol.
4
Synthesis of Neu5Gc2en (DANA-Gc) Derivative
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Neu5Gc2en (DANA-Gc) was synthesized as previously published42 (link)-45 (link) with minor modifications. Briefly, Neu5Gc (Sigma-Aldrich) was treated with Dowex 50W-X8 (H+) resin in MeOH for 20 hours at 20 °C to form the methyl ester. This ester was then treated with acetic anhydride and pyridine for 42 h at 20 °C to generate the peracetylated methyl ester, which was purified by column chromatography (50:l CHCl3- MeOH). This sample was treated with TMSOTf under dry nitrogen at 0 °C in MeCN for 6 h to induce elimination. The unsaturated compound was purified by chromatography (toluene/acetone, 3:1 → 2:1). The acetyl groups were cleaved by treatment with NaOH in MeOH over 12 hours followed by neutralization with H+ resin.
5
Synthesis of Neu5Gc2en (DANA-Gc) Derivative
6
Determination of Cellular Sialic Acid Composition
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The Sia composition of cells were determined by incubating with 2 M acetic acid at 80°C for 3 h, filtration through a Microcon 10-kDa centrifugal filter (Millipore), and drying in a SpeedVac vacuum concentrator. Released Sia were derivatized with DMB (Sigma-Aldrich) for 2.5 h at 50°C (39 (link)). HPLC analysis was performed using a Dionex UltiMate 3000 system with an Acclaim C18 column (Thermo Fisher) under isocratic elution in 7% methanol, 7% acetonitrile, and 86% water. Sia standards included bovine submaxillary mucin and commercial standards for Neu5Ac and Neu5Gc (Sigma-Aldrich). Statistical analyses were performed in GraphPad Prism software (v8).
7
Quantitative Analysis of Neu5Ac and Neu5Gc
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The chemicals: Triisopropanolamine (TIP); phosphoric acid; hydrochloric acid (HCl); sodium hydroxide (NaOH) and ethanol (HPLC grade) were purchased from Sigma-Aldrich (Australia). The standard: Neu5Ac (HPLC grade, ≥97%) and Neu5Gc (HPLC grade, ≥95%) were also purchased from Sigma-Aldrich (Australia).
Filter paper (0.45 μm, 25 mm Diameter Hydrophobic PTFE) was manufactured from Aijiren Corp (Quzhou, China). All standard solutions and the mobile phase (TIP solution) were prepared with deionized water obtained from Milli-Q water system (Millipore S.A.S 67120 Molsheim France).
For cell culture, Fetal Bovine Serum (FBS), Dulbecco's Modified Eagle Medium (DMEM), Streptomycin/Penicillin and TrypLE were purchased from Gibco by life technologies (Denmark). Pre-made Phosphate buffer solution (PBS) at appropriate concentrations and pH was purchased Sigma-Aldrich (Australia).
Filter paper (0.45 μm, 25 mm Diameter Hydrophobic PTFE) was manufactured from Aijiren Corp (Quzhou, China). All standard solutions and the mobile phase (TIP solution) were prepared with deionized water obtained from Milli-Q water system (Millipore S.A.S 67120 Molsheim France).
For cell culture, Fetal Bovine Serum (FBS), Dulbecco's Modified Eagle Medium (DMEM), Streptomycin/Penicillin and TrypLE were purchased from Gibco by life technologies (Denmark). Pre-made Phosphate buffer solution (PBS) at appropriate concentrations and pH was purchased Sigma-Aldrich (Australia).
8
Fluorescent Labeling of Sialic Acids
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The 1,2-diamino-4,5-methylenedioxybenzene (DMB) reagent to introduce the fluorescent label for chromatographic detections was prepared according to the method of Hara et al. (1989) as follows: 7 mM DMB in 1.4 M acetic acid containing 0.75 M 2-mercaptoethanol and 18 mM sodium hydrosulfite. The experimental samples and standards of Neu5Ac, Neu5Gc, and KDN (Sigma Aldrich, St. Louis, MO) were filtered through a 0.22-μm Millipore membrane filter, and 50 μL of the DMB reagent was mixed with 50 μL of the filtered sample or standard (Sigma Aldrich) in the insert in a brown high-performance liquid chromatography vial (Agilent Technologies, Santa Clara, CA) to protect the samples from light. All samples were mixed by vortex and heated at 80°C for 50 min before being cooled on ice for 5 min. Immediately after the DMB derivatization, samples were quantitatively analyzed for Sia concentration by UHPLC as described below.
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