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.

该副本是利用资源的众多研究子项目之一 由NIH/NCRR资助的中心赠款提供。对该子弹的主要支持 而且，副投影的主要研究员可能是其他来源提供的 包括其他NIH来源。列出的总费用可能 代表subproject使用的中心基础架构的估计量， NCRR赠款不直接向子弹或副本人员提供的直接资金。 方法： 插科打隔离 冷冻组织在液氮中接地，并用10 mL丙酮（两次24小时）提取，一次用二乙基醚（5 mL，30分钟）萃取，并在真空下干燥。 将组织重悬于2 ml pronase消化缓冲液中（0.1 M Tris-HCl，pH 8.0，2 mM CaCl2，1％Triton X-100），加入1.6 mg prinase酶，并在55€c（1）中摇动而消化组织。 24小时后，加入第二个1.6 mg等分试样，并持续消化24小时。将酶通过加热至100»C将酶灭活15分钟。将缓冲液调节至2 mM MGCL2，添加苯并酶（100 mU），并在37€的37 h处孵育2小时。酶灭活酶（15分钟100€c）后，未抗气的组织通过中心裂解在12000 g处通过中心分解15分钟。 将上清液应用于DEAE-Sephacel柱（2 mL），用20 mL平衡缓冲液（20 mM Tris-HCl，pH 7.5，0.5，0.1 M NaCl）洗涤，并用6 mL洗脱缓冲液（20 mm Tris-HCl pH 7.5，2.5，2 m nacl）洗脱。样品在2N NaOH中用1 ml 10％（w/v）Nabh4处理，并在4€C（2）中孵育过夜。通过添加冰川酸来停止反应，直到未形成气泡并且pH中性为中性。 将样品冷冻干燥，使用PD10色谱柱（GE Healthcare）进行脱水，再次冷冻干燥，并溶解在50 l水中。 GAG裂解酶消化 将样品溶液的10-l等分试样溶解为50 mM乙酸铵，pH 7，并用10°L的适当GAG裂解酶溶液（1 U/mL）处理。所使用的酶溶液是A）软骨素酶ABC和AC，B）软骨素ABC和C）肝素酶I，II和III。将样品在37°C过夜孵育，并加热至100€持续2分钟。 SAX-HPLC SAX-HPLC使用4.6毫米Waters Spherisorb分析柱在Agilent系统上进行，使用以下梯度在25？c时进行5？M粒径： 溶剂A：2.5 mm Na-磷酸盐，pH 3.5 溶剂B：2.5 mm Na-磷酸盐，pH 3.5，1.2 m NaCl。 如所述（3），通过柱后衍生化进行检测。简而言之，从二进制HPLC泵，0.25 m NaOH的1：1混合物和1％2-氰基乙酰氨酰胺的1：1混合物以0.5 mL/min的速度添加了从色谱柱中的洗脱液。然后将洗脱液加热至10米反应线圈中，然后在30厘米冷却线圈中冷却，然后将其定向到Shimadzu荧光检测器中。激发波长为346 nm，发射波长为410 nm。