聚丙烯酰胺凝胶电泳和银染色检测甘油糖
1Department of Medicine,University of Colorado Anschutz Medical Campus, 2Medical Scientist Training Program,University of Colorado Anschutz Medical Campus, 3Department of Chemistry and Chemical Biology,Rensselaer Polytechnic Institute, 4Department of Health Sciences,Curtin University, 5Department of Medicine,Denver Health Medical Center
Summary
本报告描述了从生物样本中分离和净化硫化甘油（GAGs）的技术，以及一种聚丙烯酰胺凝胶电泳方法，以接近其大小。GAG通过静电与蛋白质的相互作用，促进组织结构并影响信号过程。GAG 聚合物长度有助于它们对认知配体的结合亲和力。
Abstract
硫化甘油（GAGs），如硫酸肝（HS）和硫酸软骨蛋白（CS），在生物体中随处可见，在各种基本生物结构和过程中起着至关重要的作用。作为聚合物，GAG 作为聚氨酯混合物存在，其中含有聚糖链，范围从 4000 Da 到 40，000 DA 不等。在这些链条中存在着硫化领域，赋予负电荷模式，促进与认知蛋白配体的正电荷残留物的相互作用。GAG 的硫化域必须足够长度，以便进行这些静电相互作用。要了解GAG在生物组织中的功能，研究者必须能够分离、净化和测量GAG的大小。本报告描述了一种实用且多功能的聚丙烯酰胺凝胶电泳技术，该技术可利用该技术解决从各种生物组织类型中分离出来的 GAG 之间的相对较小的尺寸差异。
Introduction
糖氨糖（GAGs）是线性多糖的多样化家族，是生物体中无处不在的元素，有助于许多基本的生理过程1。硫酸肝素 （HS） 和软骨素硫酸盐 （CS） 等 GAG 可能会在多糖链的不同位置硫化，从而提供负电荷的地理域。这些GAG，当系在细胞表面的蛋白质称为蛋白糖，投射到细胞外空间，并结合识别配体，允许调节两个 cis-（利甘附着在同一细胞）和跨（利甘附着在相邻细胞）信号过程2。此外，GAG还作为结构元素在组织中发挥关键作用，如球状地下室膜3、血管内皮糖碱4和肺上皮糖糖6，以及结缔组织（如软骨6）。
GAG多糖链的长度因其生物背景而有很大差异，可以通过高度复杂的酶调控系统7动态延长、切割和修改。重要的是，GAG聚合物链的长度大大有助于它们对配体的结合亲和力，并随后对它们的生物功能8，9。因此，确定内源性GAG的功能需要对其大小进行欣赏。不幸的是，与蛋白质和核酸不同，很少有现成的技术来检测和测量GAG，这在历史上导致对这一多样化多糖家族的生物作用的调查相对有限。
本文描述了如何从大多数生物组织中分离和净化GAG，并描述了如何使用聚丙烯酰胺凝胶电泳（PAGE）来评估具有相当程度特异性的分离聚合物的长度。与其他高度复杂的（通常基于质谱）的糖电方法不同，这种方法可以使用标准的实验室设备和技术。因此，这种实际方法可以扩大调查人员确定本地GAG的生物作用及其与上下文重要配体的相互作用的能力。
Protocol
根据科罗拉多大学机构动物护理和使用委员会批准的协议，本协议中分析的所有生物样本均来自小鼠。 1. 肝硫酸盐隔离 组织样本的脱皮注：脱脂是富含脂肪组织的可选步骤。 制作甲醇和二氯甲烷的1：1混合物。每个样品准备约 500 μL;较大的组织块可能需要高达 1 mL。 将每个组织样本放入一个小玻璃容器中，盖上盖子，以进行熟食。注?…
Representative Results
阿尔西安蓝用于染色硫化GAG 10：此信号通过使用随后的银色污渍 11放大。 图1 提供了银染色开发过程的视觉演示。如前所示，当开发剂穿透聚丙烯酰胺凝胶时，代表电泳分离的 GAG 的阿尔西亚蓝色信号被放大。通常，开发过程将减少银色和阿尔西亚蓝色染色的GAG在密度依赖的方式，每个频段的边缘减少第一，而更密集的染色区域在中心将?…
Discussion
GAG 在许多不同的生物过程中发挥着核心作用。硫化 GAG（如 HS 和 CS）的主要功能之一是与配体交互并绑定，这可以改变下游信号功能。GAG 与认知配体结合亲和力的一个重要决定因素是 GAG 聚合物链的长度 8、9、14。 因此，研究人员必须能够合理精确地定义从感兴趣的生物样本中分离出来的GAG链的大小。为了实用，该技术应能够…
Disclosures
The authors have nothing to disclose.
Acknowledgements
这项工作由F31 HL143873-01（WBL），R01 HL125371（RJL和EPS）资助
Materials
| Accuspin Micro17 benchtop microcentrifuge | thermoFisher Scientific | 13-100-675 | Any benchtop microcentrifuge/rotor combination capable of 14000 xG is appropriate |
| Acrylamide (solid) | thermoFisher Scientific | BP170-100 | Electrophoresis grade |
| Actinase E | Sigma Aldrich | P5147 | Protease mix from S. griseus |
| Alcian Blue 8GX (solid) | thermoFisher Scientific | AC400460100 | |
| Ammonium acetate (solid) | thermoFisher Scientific | A639-500 | Molecular biology grade |
| Ammonium hydroxide (liquid) | thermoFisher Scientific | A669S-500 | certified ACS |
| Ammonium persulfate (solid) | thermoFisher Scientific | BP179-25 | electrophoresis grade |
| Barnstead GenPure Pro Water Purification System | ThermoFisher Scientific | 10-451-217PKG | Any water deionizing/ purification system is an acceptable substitute |
| Boric acid (solid) | thermoFisher Scientific | A73-500 | Molecular biology grade |
| Bromphenol blue (solid) | thermoFisher Scientific | B392-5 | |
| Calcium acetate (solid) | ThermoFisher Scientific | 18-609-432 | Molecular biology grade |
| Calcium chloride (solid) | ThermoFisher Scientific | AC349610250 | Molecular biology grade |
| CHAPS detergent (3-((3-cholamidopropyl) dimethylammonio)-1-propanesulfonate) | ThermoFisher Scientific | 28299 | |
| Chondroitinase ABC | Sigma Aldrich | C3667 | |
| Criterion empty cassette for PAGE (1.0mm thick, 12+2 wells) | Bio-Rad | 3459901 | Any 1.0mm thick PAGE casting cassette system will suffice |
| Criterion PAGE Cell system (cell and power supply) | Bio-Rad | 1656019 | any comparable vertical gel PAGE system will work) |
| Dichloromethane (liquid) | thermoFisher Scientific | AC610931000 | certified ACS |
| EDTA disodium salt (solid) | thermoFisher Scientific | 02-002-786 | Molecular biology grade |
| Glacial acetic acid (liquid) | thermoFisher Scientific | A35-500 | Certified ACS |
| Glycine (solid) | thermoFisher Scientific | G48-500 | Electrophoresis grade |
| Heparanase I/III | Sigma Aldrich | H3917 | From Flavobacterium heparinum |
| Heparin derived decasaccharide (dp10) | galen scientific | HO10 | |
| Heparin derived hexasaccharde (dp6) | Galen scientific | HO06 | |
| Heparin derived oligosaccharide (dp20) | galen scientific | HO20 | |
| Hydrochloric acid (liquid) | thermoFisher Scientific | A466-250 | |
| Lyophilizer | Labconco | 7752020 | Any lyophilizer that can achieve -40C and 0.135 Torr will work; can also be replaced with rotational vacuum concentrator |
| Methanol (liquid) | thermoFisher Scientific | A412-500 | Certified ACS |
| Molecular Imager Gel Doc XR System | Bio-Rad | 170-8170 | Any comparable gel imaging system is an acceptable substitute |
| N,N'-methylene-bis-acrylamide (solid) | thermoFisher Scientific | BP171-25 | Electrophoresis grade |
| Phenol red (solid) | thermoFisher Scientific | P74-10 | Free acid |
| Q Mini H Ion Exchange Column | Vivapure | VS-IX01QH24 | Ion exchange column must have minimum loading volume of 0.4mL, working pH of 2-12, and selectivity for ionic groups with pKa of 11 |
| Silver nitrate (solid) | thermoFisher Scientific | S181-25 | certified ACS |
| Sodium Acetate (solid) | ThermoFisher Scientific | S210-500 | Molecular biology grade |
| Sodium chloride (solid) | thermoFisher Scientific | S271-500 | Molecular biology grade |
| Sodium hydroxide (solid) | thermoFisher Scientific | S392-212 | |
| Sucrose (solid) | thermoFisher Scientific | BP220-1 | Molecular biology grade |
| TEMED (N,N,N',N'-tetramethylenediamine) | thermoFisher Scientific | BP150-20 | Electrophoresis grade |
| Tris base (solid) | thermoFisher Scientific | BP152-500 | Molecular biology grade |
| Ultra Centrifugal filters, 0.5mL, 3000 Da molecular weight cutoff | Amicon | UFC500324 | Larger volume filter units may be used, depending on sample size. |
| Urea (solid) | ThermoFisher Scientific | 29700 | |
| Vacufuge Plus | Eppendorf | 22820001 | Any rotational vacuum concentrator will work; can be replaced with lyophilizer |
| Vacuum filter unit, single use, 0.22uM pore PES, 500mL volume | thermoFisher Scientific | 569-0020 | Alternative volumes and filter materials acceptable |
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Cite This Article
LaRiviere, W. B., Han, X., Oshima, K., McMurtry, S. A., Linhardt, R. J., Schmidt, E. P. Detection of Glycosaminoglycans by Polyacrylamide Gel Electrophoresis and Silver Staining. J. Vis. Exp. (168), e62319, doi:10.3791/62319 (2021).