Name: detection of glycosaminoglycans by polyacrylamide gel electrophoresis and silver staining
Uploaded: 2021-02-25T15:00:00
Description: Watch this Scientific Journal Video about Detection of Glycosaminoglycans by Polyacrylamide Gel Electrophoresis and Silver Staining at JoVE.com

这项工作由F31 HL143873-01（WBL），R01 HL125371（RJL和EPS）资助

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
<ol><li>组织样本的脱皮 注：脱脂是富含脂肪组织的可选步骤。<ol>
<li>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。</li>
		<li>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?...

<ol>
	<li>LaRivi&#232;re, W. B., Schmidt, E. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+pulmonary+endothelial+glycocalyx+in+ARDS:+A+critical+role+for+heparan+sulfate.">The pulmonary endothelial glycocalyx in ARDS: A critical role for heparan sulfate.</a> Current Topics in Membrane. 82, 33-52 (2018).</li><li>Haeger, S. M., Yang, Y., Schmidt, E. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heparan+sulfate+in+the+developing,+healthy,+and+injured+lung.">Heparan sulfate in the developing, healthy, and injured lung.</a> American Journal of Respiratory Cell and Molecular Biology. 55 (1), 5-11 (2016).</li><li>Morita, H., Yoshimura, A., Kimata, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+heparan+sulfate+in+the+glomerular+basement+membrane.">The role of heparan sulfate in the glomerular basement membrane.</a> Kidney International. 73 (3), 247-248 (2008).</li><li>Schmidt, E. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+pulmonary+endothelial+glycocalyx+regulates+neutrophil+adhesion+and+lung+injury+during+experimental+sepsis.">The pulmonary endothelial glycocalyx regulates neutrophil adhesion and lung injury during experimental sepsis.</a> Nature Medicine. 18 (8), 1217-1223 (2012).</li><li>Haeger, S. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epithelial+heparan+sulfate+contributes+to+alveolar+barrier+function+and+is+shed+during+lung+injury.">Epithelial heparan sulfate contributes to alveolar barrier function and is shed during lung injury.</a> American Journal of Respiratry Cell and Molecular Biology. 59 (3), 363-374 (2018).</li><li>Mankin, H. J., Lippiello, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+glycosaminoglycans+of+normal+and+arthritic+cartilage.">The glycosaminoglycans of normal and arthritic cartilage.</a> Journal of Clinical Investigation. 50 (8), 1712-1719 (1971).</li><li>Annaval, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heparan+sulfate+proteoglycans+biosynthesis+and+post+synthesis+mechanisms+combine+few+enzymes+and+few+core+proteins+to+generate+extensive+structural+and+functional+diversity.">Heparan sulfate proteoglycans biosynthesis and post synthesis mechanisms combine few enzymes and few core proteins to generate extensive structural and functional diversity.</a> Molecules. 25 (18), (2020).</li><li>Zhang, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+the+interactions+of+different+growth+factors+and+glycosaminoglycans.">Comparison of the interactions of different growth factors and glycosaminoglycans.</a> Molecules. 24 (18), (2019).</li><li>Pempe, E. H., Xu, Y., Gopalakrishnan, S., Liu, J., Harris, E. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Probing+structural+selectivity+of+synthetic+heparin+binding+to+Stabilin+protein+receptors.">Probing structural selectivity of synthetic heparin binding to Stabilin protein receptors.</a> Journal of Biological Chemistry. 287 (25), 20774-20783 (2012).</li><li>Cowman, M. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polyacrylamide-gel+electrophoresis+and+Alcian+Blue+staining+of+sulphated+glycosaminoglycan+oligosaccharides.">Polyacrylamide-gel electrophoresis and Alcian Blue staining of sulphated glycosaminoglycan oligosaccharides.</a> Biochemical Journal. 221 (3), 707-716 (1984).</li><li>M&#248;ller, H. J., Poulsen, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+method+for+silver+staining+of+glycoproteins+in+thin+sodium+dodecyl+sulfate+polyacrylamide+gels.">Improved method for silver staining of glycoproteins in thin sodium dodecyl sulfate polyacrylamide gels.</a> Analytical Biochemistry. 226 (2), 371-374 (1995).</li><li>Min, H., Cowman, M. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Combined+alcian+blue+and+silver+staining+of+glycosaminoglycans+in+polyacrylamide+gels:+Application+to+electrophoretic+analysis+of+molecular+weight+distribution.">Combined alcian blue and silver staining of glycosaminoglycans in polyacrylamide gels: Application to electrophoretic analysis of molecular weight distribution.</a> Analytical Biochemistry. 155 (2), 275-285 (1986).</li><li>Jay, G. D., Culp, D. J., Jahnke, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Silver+staining+of+extensively+glycosylated+proteins+on+sodium+dodecyl+sulfate-polyacrylamide+gels:+Enhancement+by+carbohydrate-binding+dyes.">Silver staining of extensively glycosylated proteins on sodium dodecyl sulfate-polyacrylamide gels: Enhancement by carbohydrate-binding dyes.</a> Analytical Biochemistry. 185 (2), 324-330 (1990).</li><li>Abraham, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Liposomal+prostaglandin+E1+(TLC+C-53)+in+acute+respiratory+distress+syndrome:+a+controlled,+randomized,+double-blind,+multicenter+clinical+trial.+TLC+C-53+ARDS+Study+Group.">Liposomal prostaglandin E1 (TLC C-53) in acute respiratory distress syndrome: a controlled, randomized, double-blind, multicenter clinical trial. TLC C-53 ARDS Study Group.</a> Critical Care Medicine. 27 (8), 1478-1485 (1999).</li><li>Pervin, A., al-Hakim, A., Linhardt, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Separation+of+glycosaminoglycan-derived+oligosaccharides+by+capillary+electrophoresis+using+reverse+polarity.">Separation of glycosaminoglycan-derived oligosaccharides by capillary electrophoresis using reverse polarity.</a> Analytical Biochemistry. 221 (1), 182-188 (1994).</li><li>Wang, Z., Zhang, F., Dordick, J. S., Linhardt, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+mass+characterization+of+glycosaminoglycans+with+different+degrees+of+sulfation+in+bioengineered+heparin+process+by+size+exclusion+chromatography.">Molecular mass characterization of glycosaminoglycans with different degrees of sulfation in bioengineered heparin process by size exclusion chromatography.</a> Current Analytical Chemistry. 8 (4), 506-511 (2012).</li><li>Pepi, L. E., Sanderson, P., Stickney, M., Amster, I. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Developments+in+mass+spectrometry+for+glycosaminoglycan+analysis:+A+review.">Developments in mass spectrometry for glycosaminoglycan analysis: A review.</a> Molecular and Cellular Proteomics. , 100025 (2021).</li><li>Whiteman, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+quantitative+measurement+of+Alcian+Blue-glycosaminoglycan+complexes.">The quantitative measurement of Alcian Blue-glycosaminoglycan complexes.</a> Biochemical Journal. 131 (2), 343-350 (1973).</li><li>Yuan, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+mass+dependence+of+hyaluronan+detection+by+sandwich+ELISA-like+assay+and+membrane+blotting+using+biotinylated+hyaluronan+binding+protein.">Molecular mass dependence of hyaluronan detection by sandwich ELISA-like assay and membrane blotting using biotinylated hyaluronan binding protein.</a> Glycobiology. 23 (11), 1270-1280 (2013).</li></ol>

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...

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。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Accuspin Micro17 benchtop microcentrifuge</td>
			<td>thermoFisher Scientific</td>
			<td>13-100-675</td>
			<td>Any benchtop microcentrifuge/rotor combination capable of 14000 xG is appropriate</td>
		</tr>
		<tr>
			<td>Acrylamide (solid)</td>
			<td>thermoFisher Scientific</td>
			<td>BP170-100</td>
			<td>Electrophoresis grade</td>
		</tr>
		<tr>
			<td>Actinase E</td>
			<td>Sigma Aldrich</td>
			<td>P5147</td>
			<td>Protease mix from S. griseus</td>
		</tr>
		<tr>
			<td>Alcian Blue 8GX (solid)</td>
			<td>thermoFisher Scientific</td>
			<td>AC400460100</td>
			<td></td>
		</tr>
		<tr>
			<td>Ammonium acetate (solid)</td>
			<td>thermoFisher Scientific</td>
			<td>A639-500</td>
			<td>Molecular biology grade</td>
		</tr>
		<tr>
			<td>Ammonium hydroxide (liquid)</td>
			<td>thermoFisher Scientific</td>
			<td>A669S-500</td>
			<td>certified ACS</td>
		</tr>
		<tr>
			<td>Ammonium persulfate (solid)</td>
			<td>thermoFisher Scientific</td>
			<td>BP179-25</td>
			<td>electrophoresis grade</td>
		</tr>
		<tr>
			<td>Barnstead GenPure Pro Water Purification System</td>
			<td>ThermoFisher Scientific</td>
			<td>10-451-217PKG</td>
			<td>Any water deionizing/ purification system is an acceptable substitute</td>
		</tr>
		<tr>
			<td>Boric acid (solid)</td>
			<td>thermoFisher Scientific</td>
			<td>A73-500</td>
			<td>Molecular biology grade</td>
		</tr>
		<tr>
			<td>Bromphenol blue (solid)</td>
			<td>thermoFisher Scientific</td>
			<td>B392-5</td>
			<td></td>
		</tr>
		<tr>
			<td>Calcium acetate (solid)</td>
			<td>ThermoFisher Scientific</td>
			<td>18-609-432</td>
			<td>Molecular biology grade</td>
		</tr>
		<tr>
			<td>Calcium chloride (solid)</td>
			<td>ThermoFisher Scientific</td>
			<td>AC349610250</td>
			<td>Molecular biology grade</td>
		</tr>
		<tr>
			<td>CHAPS detergent (3-((3-cholamidopropyl) dimethylammonio)-1-propanesulfonate)</td>
			<td>ThermoFisher Scientific</td>
			<td>28299</td>
			<td></td>
		</tr>
		<tr>
			<td>Chondroitinase ABC</td>
			<td>Sigma Aldrich</td>
			<td>C3667</td>
			<td></td>
		</tr>
		<tr>
			<td>Criterion empty cassette for PAGE (1.0mm thick, 12+2 wells)</td>
			<td>Bio-Rad</td>
			<td>3459901</td>
			<td>Any 1.0mm thick PAGE casting cassette system will suffice</td>
		</tr>
		<tr>
			<td>Criterion PAGE Cell system (cell and power supply)</td>
			<td>Bio-Rad</td>
			<td>1656019</td>
			<td>any comparable vertical gel PAGE system will work)</td>
		</tr>
		<tr>
			<td>Dichloromethane (liquid)</td>
			<td>thermoFisher Scientific</td>
			<td>AC610931000</td>
			<td>certified ACS</td>
		</tr>
		<tr>
			<td>EDTA disodium salt (solid)</td>
			<td>thermoFisher Scientific</td>
			<td>02-002-786</td>
			<td>Molecular biology grade</td>
		</tr>
		<tr>
			<td>Glacial acetic acid (liquid)</td>
			<td>thermoFisher Scientific</td>
			<td>A35-500</td>
			<td>Certified ACS</td>
		</tr>
		<tr>
			<td>Glycine (solid)</td>
			<td>thermoFisher Scientific</td>
			<td>G48-500</td>
			<td>Electrophoresis grade</td>
		</tr>
		<tr>
			<td>Heparanase I/III</td>
			<td>Sigma Aldrich</td>
			<td>H3917</td>
			<td>From Flavobacterium heparinum</td>
		</tr>
		<tr>
			<td>Heparin derived decasaccharide (dp10)</td>
			<td>galen scientific</td>
			<td>HO10</td>
			<td></td>
		</tr>
		<tr>
			<td>Heparin derived hexasaccharde (dp6)</td>
			<td>Galen scientific</td>
			<td>HO06</td>
			<td></td>
		</tr>
		<tr>
			<td>Heparin derived oligosaccharide (dp20)</td>
			<td>galen scientific</td>
			<td>HO20</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrochloric acid (liquid)</td>
			<td>thermoFisher Scientific</td>
			<td>A466-250</td>
			<td></td>
		</tr>
		<tr>
			<td>Lyophilizer</td>
			<td>Labconco</td>
			<td>7752020</td>
			<td>Any lyophilizer that can achieve -40C and 0.135 Torr will work; can also be replaced with rotational vacuum concentrator</td>
		</tr>
		<tr>
			<td>Methanol (liquid)</td>
			<td>thermoFisher Scientific</td>
			<td>A412-500</td>
			<td>Certified ACS</td>
		</tr>
		<tr>
			<td>Molecular Imager Gel Doc XR System</td>
			<td>Bio-Rad</td>
			<td>170-8170</td>
			<td>Any comparable gel imaging system is an acceptable substitute</td>
		</tr>
		<tr>
			<td>N,N&#39;-methylene-bis-acrylamide (solid)</td>
			<td>thermoFisher Scientific</td>
			<td>BP171-25</td>
			<td>Electrophoresis grade</td>
		</tr>
		<tr>
			<td>Phenol red (solid)</td>
			<td>thermoFisher Scientific</td>
			<td>P74-10</td>
			<td>Free acid</td>
		</tr>
		<tr>
			<td>Q Mini H Ion Exchange Column</td>
			<td>Vivapure</td>
			<td>VS-IX01QH24</td>
			<td>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</td>
		</tr>
		<tr>
			<td>Silver nitrate (solid)</td>
			<td>thermoFisher Scientific</td>
			<td>S181-25</td>
			<td>certified ACS</td>
		</tr>
		<tr>
			<td>Sodium Acetate (solid)</td>
			<td>ThermoFisher Scientific</td>
			<td>S210-500</td>
			<td>Molecular biology grade</td>
		</tr>
		<tr>
			<td>Sodium chloride (solid)</td>
			<td>thermoFisher Scientific</td>
			<td>S271-500</td>
			<td>Molecular biology grade</td>
		</tr>
		<tr>
			<td>Sodium hydroxide (solid)</td>
			<td>thermoFisher Scientific</td>
			<td>S392-212</td>
			<td></td>
		</tr>
		<tr>
			<td>Sucrose (solid)</td>
			<td>thermoFisher Scientific</td>
			<td>BP220-1</td>
			<td>Molecular biology grade</td>
		</tr>
		<tr>
			<td>TEMED (N,N,N&#39;,N&#39;-tetramethylenediamine)</td>
			<td>thermoFisher Scientific</td>
			<td>BP150-20</td>
			<td>Electrophoresis grade</td>
		</tr>
		<tr>
			<td>Tris base (solid)</td>
			<td>thermoFisher Scientific</td>
			<td>BP152-500</td>
			<td>Molecular biology grade</td>
		</tr>
		<tr>
			<td>Ultra Centrifugal filters, 0.5mL, 3000 Da molecular weight cutoff</td>
			<td>Amicon</td>
			<td>UFC500324</td>
			<td>Larger volume filter units may be used, depending on sample size.&#160;</td>
		</tr>
		<tr>
			<td>Urea (solid)</td>
			<td>ThermoFisher Scientific</td>
			<td>29700</td>
			<td></td>
		</tr>
		<tr>
			<td>Vacufuge Plus</td>
			<td>Eppendorf</td>
			<td>22820001</td>
			<td>Any rotational vacuum concentrator will work; can be replaced with lyophilizer</td>
		</tr>
		<tr>
			<td>Vacuum filter unit, single use, 0.22uM pore PES, 500mL volume</td>
			<td>thermoFisher Scientific</td>
			<td>569-0020</td>
			<td>Alternative volumes and filter materials acceptable</td>
		</tr>
	</tbody>
</table>

detection of glycosaminoglycans by polyacrylamide gel electrophoresis and silver staining

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。

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?...

Watch this Scientific Journal Video about Detection of Glycosaminoglycans by Polyacrylamide Gel Electrophoresis and Silver Staining at JoVE.com

Detection of Glycosaminoglycans by Polyacrylamide Gel Electrophoresis and Silver Staining

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。

聚丙烯酰胺凝胶电泳和银染色检测甘油糖

Sulfated glycosaminoglycans (GAGs) such as heparan sulfate (HS) and chondroitin sulfate (CS) are ubiquitous in living organisms and play a critical role ...

该协议描述了一个简单的技术，可用于检测存在和测量糖糖糖的长度，这是分子越来越被公认为对许多生物过程的重要性。这项技术很容易适应大多数生命科学实验室。可比的糖化学技术通常需要设备和专业知识，仅在糖化学研究实验室找到。这项技术对于有兴趣研究糖合金的研究人员可能特别有用，糖合物是一种多糖涂层，与人体的内皮和上皮表面相线。首先将空盒式磁带放入 PAGE 储罐中。将 10 毫升溶胶溶液与 60 微升新鲜制备的 10%铵说服剂混合在 15 毫升管中，从而铸造溶解凝胶。然后添加 10 微升的 TEMED。轻轻倒置管子两到三次。使用移液器快速将激活的聚丙烯酰胺凝胶溶液的 10 毫升添加到盒式磁带中。用两毫升去离子过滤水覆盖，让解析凝胶聚合30分钟。解决凝胶完全聚合后，丢弃覆盖的水。将三毫升堆叠凝胶溶液与 90 微升新制备的 10%铵说服剂混合在 15 毫升管中，从而铸造堆叠凝胶。然后加入三个TEMED微升，轻轻倒置管子两到三次。使用移液器快速将堆叠凝胶溶液添加到凝固的溶液上，直到盒式磁带填充到边缘。将梳子与空聚丙烯酰胺凝胶盒一起完全插入，让堆叠凝胶聚合 30 分钟。凝胶聚合后，从盒式磁带底部取下胶带条，并将盒式磁带放回 PAGE 罐装配件中。为上下腔填充相应的缓冲解决方案。将干燥的甘油糖样品溶解在最小必要量的去离子过滤水中，并以一比一的比例与样品装载缓冲器混合。将样品和 HS 寡糖梯子加载到凝胶中。在 100 伏特下预润凝胶 5 分钟，然后以 200 伏特运行 20 至 100 分钟，具体取决于溶解凝胶溶液的丙烯酰胺百分比。运行完成后，拆解盒式磁带，小心地将凝胶提取到装满去离子过滤水的大型清洁容器中。然后丢弃水，在阿尔西亚蓝色染色溶液中涂上凝胶五分钟。丢弃阿尔西亚蓝色污渍，然后用去离子过滤水快速洗涤两到三次，直到大部分阿尔西亚蓝色染色液被去除。将凝胶涂在银硝酸盐染色溶液中，在新鲜清洁的容器中涂上 30 分钟。然后在去离子过滤水中洗两到三次，每次30分钟，以完全去除银渍溶液。丢弃水并添加开发解决方案。仔细观察凝胶的带子的外观。根据污渍的质量和加载样品的质量，开发可能需要几秒钟到几分钟的时间。一旦所需的频带可见，立即丢弃开发中的解决方案，并使用 STOP 溶液短暂清洗凝胶。分离和纯化的糖氨酸通过聚丙烯酰胺凝胶电泳在密度依赖分辨率后，使用阿尔西安蓝色和银色染色技术进行可视化。如此图所见，使用基于 PAGE 的方法，可以很容易地检测出不同聚合物长度的硫酸硫酸寡糖。这种方法的检测极限低至0.5微克，表明该技术在检测从生物样品中分离和纯化的糖方面具有高度敏感性。在使用这种方法可视化的四种不同的硫酸硫酸寡糖中，未脱损的肝素是最不容易检测到的。由于未违规肝素的多重分散性，此样品中每个单个带的密度低于纯肝素寡糖质量相等的频段。使用从小鼠身上采集的支气管虫洗液样本评估了液体生物样品中糖的纯化效率，这些样本最初含有相对较低的糖糖糖。此外，该技术的成功还利用从整个肺同质酸盐中分离出来的硫酸肝素，产生了大量分离的硫酸肝素，最小的片段质量相当于大约10个脱糖亚单位。使用新制备的试剂并过滤此协议中使用的每种试剂非常重要。使用最高纯度和质量的试剂成功执行此技术至关重要。

Research

JoVE Journal

Biology

Denaturing Gradient Gel Electrophoresis (DGGE)

Denaturing Gradient Gel Electrophoresis DGGE

变性梯度凝胶电泳（DGGE）

科研

生物学

Screening for Amyloid Aggregation by Semi-Denaturing Detergent-Agarose Gel Electrophoresis

半变性洗涤剂琼脂糖凝胶电泳筛选淀粉样蛋白聚合

Amyloid aggregation is associated with numerous protein misfolding pathologies and underlies the infectious properties of prions, which are ...

Fast and Sensitive Colloidal Coomassie G-250 Staining for Proteins in Polyacrylamide Gels

快速，灵敏的胶体考马斯亮G - 250染色蛋白聚丙烯酰胺凝胶

Coomassie Brilliant Blue (CBB) is a dye commonly used for the visualization of proteins separated by SDS-PAGE, offering a simple staining procedure and ...

Denaturing Urea Polyacrylamide Gel Electrophoresis (Urea PAGE)

Denaturing Urea Polyacrylamide Gel Electrophoresis Urea PAGE

变性尿素聚丙烯酰胺凝胶电泳（尿素页）

Urea PAGE or denaturing urea polyacrylamide gel electrophoresis employs 6-8 M urea, which denatures secondary DNA or RNA structures and is used for their ...

Blue Native Polyacrylamide Gel Electrophoresis (BN-PAGE) for Analysis of Multiprotein Complexes from Cellular Lysates

Blue Native Polyacrylamide Gel Electrophoresis BN-PAGE for Analysis of Multiprotein Complexes from Cellular Lysates

蓝原生聚丙烯酰胺凝胶电泳（BN - PAGE）分析，从细胞裂解液多蛋白复合物

Multiprotein complexes (MPCs) play a crucial role in cell signalling, since most proteins can be found in functional or regulatory complexes with other ...

Detection of Functional Matrix Metalloproteinases by Zymography

酶谱法检测功能的基质金属蛋白酶

Matrix metalloproteinases (MMPs) are zinc-containing endopeptidases. They degrade proteins by cleavage of peptide bonds. More than twenty MMPs have been ...

Pouring and Running a Protein Gel by reusing Commercial Cassettes

浇注和运行的一种蛋白质凝胶重用商​​业磁带

The evaluation of proteins using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis is a common technique used by biochemistry ...

Agarose Gel Electrophoresis for the Separation of DNA Fragments

琼脂糖凝胶电泳分离DNA片段

Agarose gel electrophoresis is the most effective way of separating DNA fragments of varying sizes ranging from 100 bp to 25 kb1. Agarose is isolated from ...

Detection of Live Escherichia coli O157:H7 Cells by PMA-qPCR

Detection of Live Escherichia coli O157:H7 Cells by PMA-qPCR

检测现场的<em&gt;大肠杆菌</em&gt; O157：H7细胞的PMA-qPCR的

A unique open reading frame (ORF) Z3276 was identified as a specific genetic marker for E. coli O157:H7. A qPCR assay was developed for detection of E. ...

Preparation of DNA-crosslinked Polyacrylamide Hydrogels

DNA交联聚丙烯酰胺水凝胶的制备

Mechanobiology is an emerging scientific area that addresses the critical role of physical cues in directing cell morphology and function. For example, ...