Name: immunoglobulin g n-glycan analysis by ultra-performance liquid chromatography
Uploaded: 2020-01-18T12:00:00
Description: Watch this Scientific Journal Video about Immunoglobulin G N-Glycan Analysis by Ultra-Performance Liquid Chromatography at JoVE.com

这项工作得到了中国国家自然科学基金（81673247和81872682）和澳大利亚-中国合作赠款（NH&MRC - APP1112767-NSFC 8156128020）的资助。

协议中的所有科目均已获首都医科大学道德委员会批准。在研究开始时，每个科目都获得了书面知情同意。
1. IgG隔离
<ol><li>制备化学品包括结合缓冲液（磷酸盐缓冲盐水，PBS）：1x PBS（pH = 7.4），中和缓冲液：10x PBS（pH = 6.6-6.8），脱液：0.1M甲酸（pH = 2.5），中和溶液的增色剂：1M碳酸氢铵，储存缓冲液：20%乙醇 = 20 mM Tris = 0.1 M NaCl （pH = 7.4），蛋白质 G 的?...

<ol>
	<li>Kolarich, D., Lepenies, B., Seeberger, P. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glycomics,+glycoproteomics+and+the+immune+system.">Glycomics, glycoproteomics and the immune system.</a> Current Opinion in Chemical Biology. 16, 214 (2012).</li><li>Cummings, R., Pierce, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Challenge+and+Promise+of+Glycomics.">The Challenge and Promise of Glycomics.</a> Chemistry Biology. 21 (1), (2014).</li><li>Shade, K. T. C., Anthony, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antibody+Glycosylation+and+Inflammation.">Antibody Glycosylation and Inflammation.</a> Antibodies. 2, 392 (2013).</li><li>Gudelj, I., Lauc, G., Pezer, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunoglobulin+G+glycosylation+in+aging+and+diseases.">Immunoglobulin G glycosylation in aging and diseases.</a> Cellular Immunology. 333, 65 (2018).</li><li>Huffman, J. 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=Comparative+performance+of+four+methods+for+high-throughput+glycosylation+analysis+of+immunoglobulin+G+in+genetic+and+epidemiological+research.">Comparative performance of four methods for high-throughput glycosylation analysis of immunoglobulin G in genetic and epidemiological research.</a> Molecular Cellular Proteomics. 13, 1598 (2014).</li><li>Stockmann, H., Adamczyk, B., Hayes, J., Rudd, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Automated,+high-throughput+IgG-antibody+glycoprofiling+platform.">Automated, high-throughput IgG-antibody glycoprofiling platform.</a> Analytical Chemistry. 85, 8841 (2013).</li><li>Kristic, J., 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=Glycans+are+a+novel+biomarker+of+chronological+and+biological+ages.">Glycans are a novel biomarker of chronological and biological ages.</a> Journals of Gerontology. Series A, Biological Sciences Medical Sciences. 69, 779 (2014).</li><li>Nikolac, P. 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=The+association+between+galactosylation+of+immunoglobulin+G+and+body+mass+index.">The association between galactosylation of immunoglobulin G and body mass index.</a> Progress in Neuropsychopharmacology Biological Psychiatry. 48, 20 (2014).</li><li>Liu, D., 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+changes+of+immunoglobulin+G+N-glycosylation+in+blood+lipids+and+dyslipidaemia.">The changes of immunoglobulin G N-glycosylation in blood lipids and dyslipidaemia.</a> Journal of Translational Medicine. 16, 235 (2018).</li><li>Lemmers, R., 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=IgG+glycan+patterns+are+associated+with+type+2+diabetes+in+independent+European+populations.">IgG glycan patterns are associated with type 2 diabetes in independent European populations.</a> Biochimica Biophysica Acta General Subjects. 1861, 2240 (2017).</li><li>Wang, Y., 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+Association+Between+Glycosylation+of+Immunoglobulin+G+and+Hypertension:+A+Multiple+Ethnic+Cross-Sectional+Study.">The Association Between Glycosylation of Immunoglobulin G and Hypertension: A Multiple Ethnic Cross-Sectional Study.</a> Medicine (Baltimore). 95, e3379 (2016).</li><li>Liu, D., 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=Ischemic+stroke+is+associated+with+the+pro-inflammatory+potential+of+N-glycosylated+immunoglobulin+G.">Ischemic stroke is associated with the pro-inflammatory potential of N-glycosylated immunoglobulin G.</a> Journal of Neuroinflammation. 15, 123 (2018).</li><li>Russell, A. C., 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+N-glycosylation+of+immunoglobulin+G+as+a+novel+biomarker+of+Parkinson's+disease.">The N-glycosylation of immunoglobulin G as a novel biomarker of Parkinson's disease.</a> GLYCOBIOLOGY. 27, 501 (2017).</li><li>Bones, J., Mittermayr, S., O'Donoghue, N., Guttman, A., Rudd, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultra+performance+liquid+chromatographic+profiling+of+serum+N-glycans+for+fast+and+efficient+identification+of+cancer+associated+alterations+in+glycosylation.">Ultra performance liquid chromatographic profiling of serum N-glycans for fast and efficient identification of cancer associated alterations in glycosylation.</a> Analytical Chemistry. 82, 10208 (2010).</li><li>Pucic, 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=High+throughput+isolation+and+glycosylation+analysis+of+IgG-variability+and+heritability+of+the+IgG+glycome+in+three+isolated+human+populations.">High throughput isolation and glycosylation analysis of IgG-variability and heritability of the IgG glycome in three isolated human populations.</a> Molecular Cellular Proteomics. 10, M111 (2011).</li><li>Berruex, L. G., Freitag, R., Tennikova, T. B. <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+antibody+binding+to+immobilized+group+specific+affinity+ligands+in+high+performance+monolith+affinity+chromatography.">Comparison of antibody binding to immobilized group specific affinity ligands in high performance monolith affinity chromatography.</a> Journal of Pharmaceutical and Biomedical Analysis. 24, 95 (2000).</li><li>Ren, S., 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=Distribution+of+IgG+galactosylation+as+a+promising+biomarker+for+cancer+screening+in+multiple+cancer+types.">Distribution of IgG galactosylation as a promising biomarker for cancer screening in multiple cancer types.</a> Cell Research. 26, 963 (2016).</li></ol>

UPLC作为相对定量分析方法5，15。结果表明，UPLC是一种稳定的检测技术，具有良好的可重复性和相对定量精度。每个峰值中的甘油量以 UPLC（相对值）的总集成面积的百分比表示。相对定量提高了测试样品的可比性。此外，96孔蛋白G板用于一次用96个样品来纯化IgG，用于高通量检测。蛋白质G结合IgG的能力大于蛋白质A的能力，如先前研究15、1...

人类蛋白质的N-糖基化是一种常见和必要的转化后修饰1，有助于相对准确地预测疾病的发生和发展。由于其结构的复杂性，预计将有超过5000个甘油结构，为疾病2提供诊断和预测生物标志物的巨大潜力。附于免疫球蛋白G（IgG）的N-甘油已被证明对IgG的功能至关重要，IgG N-糖基化参与亲炎和抗炎系统之间的平衡3。差异IgG N-糖基化参与疾病发展和进展，代表疾病病理学中涉及的易感性和功能机制。IgG N-糖基化的炎症作用与衰老、炎症性疾病、自身免疫性疾病和癌症4相关。
随着检测技术的发展， 以下方法在高通量胶质中应用最广泛：亲水相互作用色谱（HILIC）具有荧光检测（UPLC-FLR）的高性能液相色谱，用激光诱导荧光的多路毛细管凝胶电泳 电感检测（xCGE-LIF）、矩阵辅助激光解吸/电光飞行质谱仪（MALDI-TOF-MS）和液相色谱电喷雾质谱（LC-ESI-MS）.这些方法克服了以前低通量、结果不稳定、灵敏度特异性差等缺点。
UPLC被广泛用于探索IgG N-糖基化与某些疾病（即衰老7、肥胖8、血脂异常9、II型糖尿病10、高血压11、缺血性中风12和帕金森病13）之间的联系。与上述其他三种方法相比，UPLC 具有以下优势5、14。首先，它提供了一种相对定量分析方法，涉及总面积规范化的数据分析提高了每个样本的可比性。其次，设备成本和所需的专业知识相对较低，这使得实施和将糖基化生物标志物转化为临床应用变得更加容易。这里介绍UPLC，以便它可以更广泛地使用。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>2-aminobenzamide, 2-AB</td>
			<td>Sigma, China</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>96-well collection plate</td>
			<td>AXYGEN</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>96-well filter plate</td>
			<td>Pol</td>
			<td>0.45 um GHP</td>
			<td></td>
		</tr>
		<tr>
			<td>96-well monolithic plate</td>
			<td>BIA Separations</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>96-well plate rotor</td>
			<td>Eppendorf Co., Ltd, Germany</td>
			<td>T_1087461900</td>
			<td></td>
		</tr>
		<tr>
			<td>Acetic acid</td>
			<td>Sigma, China</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Acetonitrile</td>
			<td>Huihai Keyi Technology Co., Ltd, China</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Ammonium bicarbonate</td>
			<td>Shenggong Biological Engineering Co., Ltd, China</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Ammonium formate</td>
			<td>Beijing Minruida Technology Co., Ltd.</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Constant shaking incubator/rocker</td>
			<td>Zhicheng analytical instrument manufacturing co., Ltd, China</td>
			<td>ZWY-10313</td>
			<td></td>
		</tr>
		<tr>
			<td>Dextran Calibration Ladder/Glycopeptide column</td>
			<td>Watts technology Co., Ltd, China</td>
			<td>BEH column</td>
			<td></td>
		</tr>
		<tr>
			<td>Dimethyl sulfoxide (DMSO)</td>
			<td>Sigma, China</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Disodium phosphate</td>
			<td>Shenggong Biological Engineering Co., Ltd, China</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Electric ovens</td>
			<td>Tester instruments Co., Ltd</td>
			<td>202-2AB</td>
			<td></td>
		</tr>
		<tr>
			<td>Empower 3.0</td>
			<td>Waters technology Co., Ltd, America</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Huihai Keyi Technology Co., Ltd, China</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Formic acid</td>
			<td>Sigma, China</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>GlycoProfile 2-AB Labeling kit</td>
			<td>Sigma, China</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>HCl</td>
			<td>Junrui Biotechnology Co., Ltd, China</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>High-speed centrifuge</td>
			<td>Eppendorf Co., Ltd, Germany</td>
			<td>5430</td>
			<td></td>
		</tr>
		<tr>
			<td>Igepal</td>
			<td>Sigma, China</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Low temperature centrifuge</td>
			<td>Eppendorf Co., Ltd, Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Low temperature refrigerator</td>
			<td>Qingdao Haier Co., Ltd</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Manifold 96-well plate</td>
			<td>Watts technology Co., Ltd, China</td>
			<td>186001831</td>
			<td></td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>Huihai Keyi Technology Co., Ltd, China</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Milli-Q pure water meter</td>
			<td>Millipore Co., Ltd, America</td>
			<td>Advantage A10</td>
			<td></td>
		</tr>
		<tr>
			<td>NaOH</td>
			<td>Shenggong Biological Engineering Co., Ltd, China</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>PH tester</td>
			<td>Sartorius Co., Ltd, Germany</td>
			<td>PB-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate buffered saline, PBS</td>
			<td>Shenggong Biological Engineering Co., Ltd, China</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Pipette</td>
			<td>Eppendorf Co., Ltd, Germany</td>
			<td>4672100, 0.5-10&#956;l &#38; 10-100&#956;l &#38; 20-200&#956;l &#38; 1000&#956;l</td>
			<td></td>
		</tr>
		<tr>
			<td>PNGase F enzyme</td>
			<td>Sigma, China</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium dihydrogen phosphate</td>
			<td>Shenggong Biological Engineering Co., Ltd, China</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Propan-2-ol</td>
			<td>Huihai Keyi Technology Co., Ltd, China</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>SDS</td>
			<td>Sigma, China</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium chloride</td>
			<td>Shenggong Biological Engineering Co., Ltd, China</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium cyanoborohydride (NaBH3CN)</td>
			<td>Sigma, China</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Spectrophotometer</td>
			<td>Shanghai Yuanxi instrument Co., Ltd</td>
			<td>B-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Transfer liquid gun</td>
			<td>Smer Fell Science and Technology Co., Ltd, China</td>
			<td>4672100</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris</td>
			<td>Amresco, America</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Ultra-low temperature refrigerator</td>
			<td>Thermo Co., Ltd, America</td>
			<td>MLT-1386-3-V; MDF-382E</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultra-performance liquid chromatography</td>
			<td>Watts technology Co., Ltd, China</td>
			<td>Acquity MLtraPerformance LC</td>
			<td></td>
		</tr>
		<tr>
			<td>Vacuum Pump</td>
			<td>Watts technology Co., Ltd, China</td>
			<td>725000604</td>
			<td></td>
		</tr>
		<tr>
			<td>Volatilizing machine/Dryer</td>
			<td>Eppendorf Co., Ltd, Germany</td>
			<td>T_1087461900</td>
			<td></td>
		</tr>
		<tr>
			<td>Vortex</td>
			<td>Changzhou Enpei instrument Co., Ltd, China</td>
			<td>NP-30S</td>
			<td></td>
		</tr>
		<tr>
			<td>Water-bath</td>
			<td>Tester instruments Co., Ltd</td>
			<td>DK-98-IIA</td>
			<td></td>
		</tr>
		<tr>
			<td>Weighing balance</td>
			<td>Shanghai Jingke Scientific Instrument Co., Ltd.</td>
			<td>MP200B</td>
			<td></td>
		</tr>
	</tbody>
</table>

immunoglobulin g n-glycan analysis by ultra-performance liquid chromatography

Glycomics 是组学系统研究的一个新的子专业，在发现下一代疾病易感性、药物靶点发现和精密医学的生物标志物方面具有巨大潜力。替代IgG N-甘油已报告在几个常见的慢性疾病，并建议在临床应用（即，诊断和预测疾病的生物标志物）有很大的潜力。IgG N-甘油采用亲水相互作用色谱法（HILIC）超高性能液相色谱（UPLC）进行广泛描述。UPLC是一种稳定的检测技术，具有良好的可重复性和较高的相对定量精度。此外，IgG N-甘甘的结构明显分离，血浆中的甘油成分和相对丰度具有特征。

如图1所示，IgG N-甘油根据峰值位置和保留时间分析为24初始IgG甘油峰（GP）。根据先前的研究（表1）15，N-甘油结构可通过质谱检测获得。 为了确保结果具有可比性，应用了总面积正常化，其中每个峰值中的甘油量以总综合面积的百分比表示。
为了评估该方法?...

Watch this Scientific Journal Video about Immunoglobulin G N-Glycan Analysis by Ultra-Performance Liquid Chromatography at JoVE.com

Immunoglobulin G N-Glycan Analysis by Ultra-Performance Liquid Chromatography

免疫球蛋白G（IgG）N-甘油采用亲水相互作用色谱UPLC的特征是。 此外，IgG N-甘油的结构明显分离。这里介绍这个实验方法，以便它可以在研究环境中广泛使用。

免疫球蛋白 G N-甘油分析通过超高性能液色谱分析

Glycomics is a new subspecialty in omics system research that offers significant potential in discovering next-generation biomarkers for disease ...

超高性能液相色谱是建立对IgG n-甘油进行精确分析的技术，由于其灵敏度，并且具有提供甘油特定信息的能力。血浆中蛋白质的测量可能很吸引人。在医学。也与糖基中的 IgG 和含有种群的此类和生物相关。你知道不纯状态用于测试甘油中的 IgG。实际上，它可以测试蛋白质的n-甘丹。这需要在两种纯状态的交界蛋白中应用于蛋白质 OR 实验过程相对简单，而实验过程需要标准化。端到端的实验过程需要三天时间才能操作，实验步骤需要记忆才能掌握实验技能。要准备样品，请解冻冷冻血浆样品。然后，在80倍G下离心10分钟。将蛋白质G单片板留在室温下30分钟。将 100 微米的样品转移到两毫升收集板的每个井中。将超纯水添加为一个控制样品。用一个 X PBS 按体积比稀释样品 1 到 7 个体积。用 200 微升超纯水清洁 0.45 微米亲水多丙烯滤板。重复清洁两次。将稀释的样品转移到过滤板中，并使用压力为 266.6 至 399.9 Pascal 的真空泵将样品过滤到集体板中。要准备蛋白质G单片板，首先丢弃储存缓冲液。用两毫升超纯水清洁单片板，一个 X PBS 的两毫升，一毫升 0.17 摩尔甲酸，两毫升 10 X PBS，以及两毫升一个 X PBS 的两毫升。使用真空泵清除以下液体。使用多通道移液器，将过滤的样品转移到蛋白质 G 单片板中，用于 IgG 结合和清洁。然后，添加两毫升一个 X PBS 来清洁单片板。使用真空泵拆下 PBS，再重复清洁两次。用一毫升0.17摩尔甲酸过滤IgG，用真空泵将样品过滤到收集板中。然后，在收集板中加入170微升的一个碳酸氢铵摩尔铵。使用 280 纳米最佳波长的吸收光谱仪检测 IgG 浓度。将提取的IgG放入烤箱中，在60摄氏度下干燥3小时。根据手稿中描述的浓度确定要观测的量，并在零下 80 摄氏度中保存。收集干燥的IgG，并储存化学品，包括1.33%SDS、4%的IgGePal和5%的PBS在室温下。用250微升超纯水稀释250单位酶，以准备内多利糖酶F酶。要使 IgG 变性，请添加 30 微升 1.33%SDS，然后通过涡流混合。将样品放入65摄氏度的烤箱中10分钟。然后，将其从烤箱中取出，让它冷却15分钟。将 10 微升 4% 的 IgePal 添加到同一样本中，并将其放在摇晃的培养箱上 5 分钟。加入 20 微升 5 X PBS 和 30 至 35 微升 0.1 摩尔氢氧化钠，将 pH 调节至 8.0，然后通过涡旋混合。加入四微升的内糖糖酶F酶，通过涡流混合。然后，在37摄氏度的水浴中孵育18至20小时。第二天，将释放的陀螺放在60摄氏度的烤箱中晾干两个半小时到三个小时。将释放甘油保存在零下80摄氏度，直到进一步测量。使用 24.5 微升的 DMSO、10.5 微升醋酸、0.7 毫克的 2 个氨基苯甲胺和 6 毫克氰酸氢钠准备两种氨基苯甲酰胺标记试剂。接下来，在黑暗的房间里，添加两个氨基苯胺标签试剂的35微升，给甘油贴上标签。将标记的 glcan 转移到振荡器 5 分钟，然后在 65 摄氏度下将其转移到烤箱中 3 小时。之后，将其转移到室温30分钟。IgG n-糖酶，我们将用标签，以便通过荧光检测检测不纯状态。预处理0.2微米GHP滤盘，200微升70%乙醇，200微升超纯水，200微升96%亚硝酸盐在4摄氏度。然后，使用真空泵清除废物。为了净化两种标有甘油的氨基苯胺，在样品中加入700微升100%乙酰胺，并将其转移到摇摇培养箱中5分钟。在摄氏4度下以134倍G离心5分钟。将超滤板转移到 0.2 微米 GHP 滤芯板上两分钟，并使用真空去除滤芯。在4摄氏度下用200微升96%乙酰胺清洗两种标有甘油的氨基苯胺，并使用真空泵清除滤液5至6次。用100微升超纯水三次标记两个氨基苯胺。将标有甘油的两种氨基苯胺转移到烤箱中，在60摄氏度下干燥三个半小时。将标有 n 甘油的甘油保存在零下 80 摄氏度，直到进一步测量。首先，打开软件来控制移动阶段。用 50%溶剂 B 和 50%溶剂 C 清洗 UPLC 仪器，流量为每分钟 0.2 毫升，使用 30 分钟。然后，用25%的溶剂 A 和 75%的溶剂 B 以每分钟 0.2 毫升的流速洗涤 20 分钟。然后，添加每分钟 0.4 毫升的流速。以 2 比 1 体积比的体积，用 25 微升的混合物（100%乙酰酸酯和超纯水）溶解标有 n-甘油的甘油。然后，在4摄氏度下以134次G离心5分钟，并使用干管将10微升的超高升装入 UPLC 仪器。以每分钟 0.4 毫升的流速分离标签的 n-甘油，线性梯度为 75%至 62%乙酰三叶草 25 分钟。然后，在 60 摄氏度的 UPLC 上执行由德克斯特校准梯甘肽柱进行分析。分别检测330纳米和420纳米的X引文和发射波长的n-甘油荧光。如图所示，根据峰值位置和保留时间，将IgG n-甘油分析为24个初始IgG甘油峰值。通过质谱检测，n-甘油结构有24种类型可供选择。为了检验该方法的可重复性和稳定性，对标准样品进行了六次并行测试。比例相对较小的甘油峰表现出超过10%变异系数的高测量误差。在这件作品中，重要的是要形成IgG n-糖原结构的确立，特别是对于建立终端来说，代替3.3IgG n-糖酶的发现是IgG的重要性。甘油来自已知是不同的。随着甘油原体学的发展，结合甘油来探索信息，这样它就可以作为今天诊断的潜力。在此技术开发之后，它提供了一种新的途径来探索

Research

JoVE Journal

Medicine

Mouse Model of Intraluminal MCAO: Cerebral Infarct Evaluation by Cresyl Violet Staining

小鼠模型的腔内MCAO脑梗塞评价甲酚紫染色

Stroke is the third  cause of mortality and the leading cause of disability in the World. Ischemic  stroke accounts for approximately 80% of all strokes. ...

科研

医学

Retinal Detachment Model in Rodents by Subretinal Injection of Sodium Hyaluronate

视网膜脱离模型鼠害由透明质酸钠的视网膜下注射

Subretinal injection of sodium hyaluronate is a widely accepted method of inducing retinal detachment (RD). However, the height and duration of RD or the ...

In utero Measurement of Heart Rate in Mouse by Noninvasive M-mode Echocardiography

In utero Measurement of Heart Rate in Mouse by Noninvasive M-mode Echocardiography

心率在小鼠中通过无创M型超声心动图测量子宫内

Congenital heart disease (CHD) is the most frequent noninfectious cause of death at birth. The incidence of CHD ranges from 4 to 50/1,000 births (Disease ...

Isolation of Human Lymphatic Endothelial Cells by Multi-parameter Fluorescence-activated Cell Sorting

人淋巴管内皮细胞中的多参​​数荧光激活细胞分选分离

Lymphatic system disorders such as primary lymphedema, lymphatic malformations and lymphatic tumors are rare conditions that cause significant morbidity ...

VDJ-Seq: Deep Sequencing Analysis of Rearranged Immunoglobulin Heavy Chain Gene to Reveal Clonal Evolution Patterns of B Cell Lymphoma

VDJ-SEQ：重排免疫球蛋白重链基因的深度测序分析，以揭示的B细胞淋巴瘤克隆演变模式

Understanding tumor clonality is critical to understanding the mechanisms involved in tumorigenesis and disease progression. In addition, understanding ...

Exosomal miRNA Analysis in Non-small Cell Lung Cancer (NSCLC) Patients' Plasma Through qPCR: A Feasible Liquid Biopsy Tool

Exosomal miRNA Analysis in Non-small Cell Lung Cancer NSCLC Patients' Plasma Through qPCR: A Feasible Liquid Biopsy Tool

外来体miRNA的分析非小细胞肺癌（NSCLC）患者的血浆通过定量PCR：一种可行的液体活检工具

The discovery of alterations in the EGFR and ALK genes, amongst others, in NSCLC has driven the development of targeted-drug therapy using selective ...

Spatial Quantification of Drugs in Pulmonary Tuberculosis Lesions by Laser Capture Microdissection Liquid Chromatography Mass Spectrometry (LCM-LC/MS)

Spatial Quantification of Drugs in Pulmonary Tuberculosis Lesions by Laser Capture Microdissection Liquid Chromatography Mass Spectrometry LCM-LC/MS

激光捕获显微切割液相色谱质谱 (LCM-LC/MS) 对肺结核患者药物的空间量化

Tuberculosis is still a leading cause of morbidity and mortality worldwide. Improvements to existing drug regimens and the development of novel ...

Application of Biochip Microfluidic Technology to Detect Serum Allergen-specific Immunoglobulin E (sIgE)

Application of Biochip Microfluidic Technology to Detect Serum Allergen-specific Immunoglobulin E sIgE

生物芯片微流控技术在血清过敏源特异性免疫球蛋白 E (sIgE) 检测中的应用

Allergic disease is common in both adults and children. Identification of the causative allergens is significant in disease management and prevention. ...

Assessment of the Acute Inhalation Toxicity of Airborne Particles by Exposing Cultivated Human Lung Cells at the Air-Liquid Interface

通过在空气-液体界面中暴露培养的人类肺细胞，评估空气中颗粒的急性吸入毒性

Here, we present a specially designed modular in vitro exposure system that enables the homogenous exposure of cultivated human lung cells at the ALI to ...

Quality-Controlled Sputum Analysis by Flow Cytometry

通过流式细胞术进行质量控制痰液分析

Sputum, widely used to study the cellular content and other microenvironmental features to understand the health of the lung, is traditionally analyzed ...