GAG ISOLATION AND SAX-HPLC OF 2 SAMPLES

2 个样品的 GAG 分离和 SAX-HPLC

• 批准号: 8361838
• 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/8361838
• 关键词: Acetic Acids; Acetone; Aliquot; Buffers; Centrifugation; Chondroitin ABC Lyase; Detection; Digestion; Enzymes; Ethyl Ether; Fluorescence; Freeze Drying; Freezing; Funding; GAG Gene; Grant; Healthcare; Heating; High Pressure Liquid Chromatography; Incubated; Liquid substance; Lyase; Magnesium Chloride; Methods; National Center for Research Resources; Nitrogen; Particle Size; Principal Investigator; Pronase; Pump; Reaction; Research; Research Infrastructure; Resources; Sampling; Solutions; Solvents; Source; System; Tissues; Triton X100; United States National Institutes of Health; Vacuum; Water; ammonium acetate; benzonase; 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: GAG Isolation The frozen tissue was ground in liquid nitrogen and extracted with 10 ml acetone (twice for 24 h) and once with diethyl ether (5 ml, 30 min) and dried under vacuum. The tissue was resuspended in 2 mL pronase digestion buffer (0.1 M Tris-HCl, pH 8.0, 2 mM CaCl2, 1% Triton X-100), 1.6 mg pronase was added, and the tissue was digested with shaking at 55 ¿C (1). After 24 h, a second 1.6 mg aliquot of pronase was added and digestion continued for 24 h. The enzyme was inactivated by heating to 100 ¿C for 15 min. The buffer was adjusted to 2 mM MgCl2, benzonase (100 mU) was added, and the sample was incubated for 2 h at 37 ¿C. After inactivation of the enzyme (15 min 100 ¿C) the undigested tissue was precipitated by centrifugation for 15 min at 12000 g. The supernatant was applied to a DEAE-Sephacel column (2 mL), washed with 20 mL equilibration buffer (20 mM Tris-HCl, pH 7.5, 0.1 M NaCl), and eluted with 6 mL elution buffer (20 mM Tris-HCl pH 7.5, 2 M NaCl). The sample was treated with 1 mL 10 % (w/v) NaBH4 in 2N NaOH, and incubated overnight at 4 ¿C (2). The reaction was stopped by adding glacial acetic acid until no bubbles were formed and the pH was neutral. The sample was freeze-dried, desalted using a PD10 column (GE Healthcare), again freeze-dried, and dissolved in 50 ¿L water. GAG lyase digestion A 10-¿L aliquot of the sample solution was dissolved 50 mM ammonium acetate, pH 7, and treated with 10 ¿L of the appropriate GAG lyase solution (1 U/mL). The enzyme solutions used were a) chondroitinases ABC and AC, b) chondroitinase ABC, and c) heparinases I, II, and III. The samples were incubated at 37 ¿C overnight and heated to 100 ¿C 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 gradients: 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 (3). 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： GAG 隔离 将冷冻组织在液氮中研磨，并用10ml丙酮提取（两次，持续24小时），用乙醚提取一次（5ml，30分钟），并在真空下干燥。 将组织重悬于 2 mL 链霉蛋白酶消化缓冲液（0.1 M Tris-HCl，pH 8.0，2 mM CaCl2，1% Triton X-100）中，加入 1.6 mg 链霉蛋白酶，在 55 °C 下振荡消化组织。 C (1). 24 小时后，添加第二份 1.6 mg 链霉酶，并继续消化 24 小时，通过加热至 100 ℃ 使酶失活。 C 15 分钟，将缓冲液调整为 2 mM MgCl2，添加 Benzonase (100 mU)，并将样品在 37 °C 下孵育 2 小时。 C. 酶失活后（100°C 15 分钟），通过在 12000 g 下离心 15 分钟沉淀未消化的组织。 将上清液上样到 DEAE-Sephacel 柱 (2 mL)，用 20 mL 平衡缓冲液（20 mM Tris-HCl，pH 7.5，0.1 M NaCl）洗涤，并用 6 mL 洗脱缓冲液（20 mM Tris-HCl pH）洗脱7.5, 2 M NaCl) 样品用 1 mL 10 % (w/v) NaBH4 处理。 2N NaOH，4℃孵育过夜C(2).加入冰醋酸终止反应直至无气泡形成且pH为中性。 将样品冷冻干燥，使用 PD10 柱（GE Healthcare）脱盐，再次冷冻干燥，并溶解在 50 ¿升水。 GAG裂解酶消化 10-¿将 L 份样品溶液溶解在 50 mM 醋酸铵中，pH 7，并用 10 ¿ L 合适的 GAG 裂解酶溶液 (1 U/mL)。所用酶溶液为 a) 软骨素酶 ABC 和 AC，b) 软骨素酶 ABC，以及 c) 肝素酶 I、II 和 III。样品在 37 ℃下孵育。 ¿ C过夜并加热至100 ¿ C 2 分钟。 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 (3) 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（0.5 mL/min）。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。