DISACCHARIDE ANALYSIS BY SAX-HPLC OF 1 SAMPLE

这个子项目是利用资源的许多研究子项目之一。 由NIH/NCRR资助的中心拨款提供。对子项目的主要支持 子项目的首席调查员可能是由其他来源提供的， 包括美国国立卫生研究院的其他来源。为子项目列出的总成本可能 表示该子项目使用的中心基础设施的估计数量， 不是由NCRR赠款提供给次级项目或次级项目工作人员的直接资金。 方法： 软骨素酶ABC消化 将含有0.1 mg/mLGAG、50 mMNH4OAc缓冲液、pH为7、软骨素酶AC(A.auresens，Sigma)0.05mU/mL的L总体积的溶液在37℃下孵育18h。 SAX-高效液相色谱 采用Agilent系统，色谱柱为4.6 mm Waters Spherisorb，粒径为5 mm，柱温为25℃，检测波长为232 nm。 溶剂A：2.5 mM磷酸二氢钠，pH 3.5 溶剂B：2.5 mM磷酸二氢钠，pH 3.5，1.2M氯化钠。 采用柱后衍生化方法进行检测。简单地说，将0.25M氢氧化钠和1%2-氰基乙酰胺的1：1混合物加入到柱上的洗脱液中，以0.5mL/min的速度从二元高效液相色谱泵中加入。洗脱液在10米的反应线圈中加热到120℃，然后在50厘米的冷却线圈中冷却，并直接进入岛津荧光检测器。激发波长为346 nm，发射波长为410 nm。

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: Chondroitinase ABC digestion A solution (100 ¿L total volume) containing 0.1 mg/mL GAG, 50 mM NH4OAc buffer, pH 7, and 0.05 mU/mL chondroitinase AC (A. aurescens, Sigma) was incubated at 37 ¿C for 18 h. 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. Analytes were detected by their UV absorbance at 232 nm using the following system. 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. 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 50-cm cooling coil, and directed into a Shimadzu fluorescence detector. Excitation wavelength was 346 nm and emission wavelength was 410 nm.