Midura Ronald J, Cali Valbona, Lauer Mark E, Calabro Anthony, Hascall Vincent C
Lerner Research Institute, Cleveland Clinic, Cleveland, OH, United States.
Lerner Research Institute, Cleveland Clinic, Cleveland, OH, United States.
Methods Cell Biol. 2018;143:297-316. doi: 10.1016/bs.mcb.2017.08.017. Epub 2017 Oct 13.
Hyaluronan (HA) exhibits numerous important roles in physiology and pathologies, and these facts necessitate an ability to accurately and reproducibly measure its quantities in tissues and cell cultures. Our group previously reported a rigorous and analytical procedure to quantify HA (and chondroitin sulfate, CS) using a reductive amination chemistry and separation of the fluorophore-conjugated, unsaturated disaccharides unique to HA and CS on high concentration acrylamide gels. This procedure is known as fluorophore-assisted carbohydrate electrophoresis (FACE) and has been adapted for the detection and quantification of all glycosaminoglycan types. While this previous FACE procedure is relatively straightforward to implement by carbohydrate research investigators, many nonglycoscience laboratories now studying HA biology might have difficulties establishing this prior FACE procedure as a routine assay for HA. To address this need, we have greatly simplified our prior FACE procedure for accurate and reproducible assessment of HA in tissues and cell cultures. This chapter describes in detail this simplified FACE procedure and, because it uses an enzyme that degrades both HA and CS, investigators will also gain additional insight into the quantities of CS in the same samples dedicated for HA analysis.
透明质酸（HA）在生理学和病理学中发挥着众多重要作用，基于这些事实，需要具备在组织和细胞培养物中准确且可重复地测量其含量的能力。我们小组之前报道了一种严谨的分析方法，该方法利用还原胺化化学以及在高浓度丙烯酰胺凝胶上分离HA和硫酸软骨素（CS）特有的荧光团共轭不饱和二糖来定量HA（和CS）。此方法被称为荧光团辅助碳水化合物电泳（FACE），并且已被用于检测和定量所有类型的糖胺聚糖。虽然之前的这种FACE方法对于碳水化合物研究人员来说实施起来相对简单，但现在许多研究HA生物学的非糖科学实验室在将之前的FACE方法确立为HA的常规检测方法时可能会遇到困难。为满足这一需求，我们对之前的FACE方法进行了大幅简化，以便在组织和细胞培养物中准确且可重复地评估HA。本章详细描述了这种简化的FACE方法，并且由于它使用了一种能同时降解HA和CS的酶，研究人员还将对用于HA分析的相同样品中的CS含量有更多了解。
