DISACCHARIDE ANALYSIS BY SAX-HPLC OF 3 SAMPLES

通过 SAX-HPLC 对 3 个样品进行二糖分析

• 批准号: 8361839
• 负责人: Parastoo Azadi
• 金额: $ 0.18万
• 依托单位: UNIVERSITY OF GEORGIA
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-02-01 至 2012-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8361839
• 关键词: Aliquot; Buffers; Detection; Digestion; Disaccharides; Fluorescence; Funding; Furuncles; GAG Gene; Grant; Heating; Heparin Lyase; High Pressure Liquid Chromatography; Incubated; Methods; National Center for Research Resources; Particle Size; Phosphate Buffer; Potassium Phosphate; Principal Investigator; Pump; Reaction; Research; Research Infrastructure; Resources; Sampling; Solutions; Solvents; Source; System; United States National Institutes of Health; Water; calcium acetate; cost; detector; inorganic phosphate

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Methods: Heparinase digestion A 20 ¿L aliquot of a 20 g/L solution of the GAG sample in water was diluted with 80 ¿L 100 mM NaOAc buffer, pH 7, containing 2 mM calcium acetate and 1 g/L BSA. The mixture was then treated with 20 ¿L of a mixture of heparinases I, II, and III (0.5 U/mL each) in 10 mM potassium phosphate buffer, pH 7, containing 2 g/L BSA and incubated at 23 ¿C. After 48 h, the reaction was quenched by boiling the mixture for 2 min. SAX-HPLC SAX-HPLC was carried out on an Agilent system using a 4.6¿250 mm Waters Spherisorb analytical column with 5 ¿m particle size at 25 ¿C using the following gradient: Solvent A: 2.5 mM Na-phosphate, pH 3.5 Solvent B: 2.5 mM Na-phosphate, pH 3.5, 1.2 M NaCl. Detection was performed by post-column derivatization as described (1). Briefly, to the eluent from the column was added, from a binary HPLC pump, a 1:1 mixture of 0.25 M NaOH and 1 % 2-cyanoacetamide at 0.5 mL/min. The eluent was then heated to 120 ¿C in a 10-m reaction coil, followed by cooling in a 30-cm cooling coil, and directed into a Shimadzu fluorescence detector. Excitation wavelength was 346 nm and emission wavelength was 410 nm.

该子项目是利用资源的众多研究子项目之一 由 NIH/NCRR 资助的中心拨款提供 该子项目的主要支持。 并且子项目的主要研究者可能是由其他来源提供的， 包括其他 NIH 来源的子项目可能列出的总成本。 代表子项目使用的中心基础设施的估计数量， NCRR 赠款不直接向子项目或子项目工作人员提供资金。 方法： 肝素酶消化 20°将 L 份 20 g/L 的 GAG 样品水溶液用 80 ¿ L 100 mM NaOAc 缓冲液，pH 7，含有 2 mM 乙酸钙和 1 g/L BSA，然后用 20 ¿ L 肝素酶 I、II 和 III（各 0.5 U/mL）混合物，溶于 10 mM 磷酸钾缓冲液（pH 7），含有 2 g/L BSA，并在 23 ℃ 下孵育48小时后，通过将混合物煮沸2分钟来猝灭反应。 SAX-HPLC SAX-HPLC 在安捷伦系统上进行，使用 4.6¿250 mm Waters Sperisorb 分析柱，具有 5¿ m 粒径（25 ¿） C 使用以下梯度： 溶剂 A：2.5mM 磷酸钠，pH 3.5 溶剂 B：2.5mM 磷酸钠，pH 3.5，1.2M NaCl。 如 (1) 中所述，通过柱后衍生化进行检测。简言之，从二元 HPLC 泵向柱洗脱液中添加 0.25 M NaOH 和 1% 2-氰基乙酰胺的 1:1 混合物，浓度为 0.5 mL/。然后将洗脱液加热至 120 ¿ C在10-m反应线圈中，随后在30-cm冷却线圈中冷却，并导入Shimadzu荧光检测器，激发波长为346 nm，发射波长为410 nm。