<html> 1。酶法deglycosylation <ol><li>使用PCR管中，以尽量减少因蒸发的水分流失。一个标签设置管1至7。 </li><li>解冻10X G7的缓冲区，10X糖蛋白变性缓冲，和10％的NP - 40，并轻轻拍打管混合的内容。保持室温。 </li><li>含酶的样品瓶置于冰上。尽量减少解冻/冻结周期。 </li><li>溶解于600μLDH 2 OhCGβ小瓶（150微克）的内容，并保持在冰上。 </li><li> DH 2 O稀释10倍的股票，准备1毫升的1X G7的缓冲区</li><li> 0.5μLPNGase F稀释25μL1X G7的缓冲区，并保持在冰上。 </li><li>结合α- N - Acetylgalactosaminidase每2μL，α1- 2岩藻糖苷酶，β1- 3半乳糖苷酶和α1- 3，6半乳糖苷酶的准备exoglycosidase组合（如混合）。 </li><li>设置PCR管所示： </li></ol>AK“&gt; <table border="1"><tbody><tr><td> 管/样品＃ </td><td> 1 </td><td> 2 </td><td> 3 </td><td> 4 </td><td> 5 </td><td> 6 </td><td> 7 </td></tr><tr><td> hCGβ（0.25mg/ml） </td><td> 9μl </td><td> 9μl </td><td> 9μl </td><td> 9μl </td><td> - </td><td> - </td><td> - </td></tr><tr><td> 10X糖蛋白变性缓冲</td><td>加入1μl </td><td>加入1μl </td><td>加入1μl </td><td>加入1μl </td><td>加入1μl </td><td>加入1μl </td><td>加入1μl </td></tr><tr><td> DH 2 O </td><td> - </td><td> - </td><td> - </td><td> - </td><td> 9μl </td><td> 9μl </td><td> 9μl </td></tr></tbody></table><ol start="9"><li>第管，轻轻混匀并放置在热循环，盖上盖子，变性的蛋白质，94孵育10分钟° C，4 ° C持有（使用PCR管在热循环，极大地防止蒸发，体积小样本）。 </li><li>删除从热循环管和离心机，以消除任何可见凝结。 </li><li>表示添加下列试剂： </li></ol><table border="1"><tbody><tr><td> 样品＃ </td><td> 1 </td><td> 2 </td><td> 3 </td><td> 4 </td><td> 5 </td><td> 6 </td><td> 7 </td></tr><tr><td> 10％NP - 40 </td><td> 2.5μl </td><td> 2.5μl </td><td> 2.5μl </td><td> 2.5μl </td><td> 2.5μl </td><td> 2.5μl </td><td> 2.5μl </td></tr><tr><td> 10X G7的缓冲区</td><td> 2.5μl </td><td> 2.5μl </td><td> 2.5μl </td><td> 2.5μl </td><td> 2.5μl </td><td> 2.5＆万亩;升</td><td> 2.5μl </td></tr><tr><td> PNGase F 1:50 DIL。 </td><td> - </td><td> 2μL </td><td> - </td><td> - </td><td> 2μL </td><td> - </td><td> - </td></tr><tr><td> Deglycosylation混合</td><td> - </td><td> - </td><td> 2μL </td><td> 2μL </td><td> - </td><td> 2μL </td><td> 2μL </td></tr><tr><td>乙二醇混合</td><td> - </td><td> - </td><td> - </td><td> 2μL </td><td> - </td><td> - </td><td> 2μL </td></tr><tr><td> DH 2 O </td><td>加入10μl </td><td> 8μl </td><td> 8μl </td><td> 6μl </td><td> 8μl </td><td> 8μl </td><td> 6μl </td></tr><tr><td> 反应总卷。 </td><td> 25μ升 </td><td> 25μ升 </td><td> 25μ升 </td><td> 25μL &lt;/ TD&gt; <td> 25μ升 </td><td> 25μ升 </td><td> 25μ升 </td></tr></tbody></table><ol start="12"><li>关闭使用新帽（丢弃用过的，因为他们不适合一个孵化周期后）的PCR管。 </li><li>混合管轻轻拍打的4倍，然后旋转内容。 </li><li>放置在热循环，并培育管在37 ° C 4小时，然后冷却至4 ° C的样品</li></ol> 2。 deglycosylated样品的SDS - PAGE <ol><li>准备加入4μL1.25M的DTT 130μL新鲜3X还原SDS上样缓冲液。 </li><li>每个样品中加入12.5μL减少准备3X SDS上样缓冲液。 </li><li>管关闭，新的警帽，并轻轻拍打管混合。 </li><li>孵育94℃，5分钟，然后冷却至4 ° C的热循环管</li><li>装入每个样品30μL和10μL在10-20％Tris -甘氨酸凝胶蛋白标记。除部分3.1样品的其余部分。加载中加入10μlColorPlus预染蛋白Marker。 </li><li> Electrophorese在室温下凝胶在130伏，直至染料前端接近凝胶底部。 </li><li>当凝胶运行完毕后，删除自投凝胶，放置在一个小塑料盒与足够的Coomasie蓝染色覆盖凝胶。 </li><li>凝胶染色，轻轻搅拌1小时。 </li><li> destain解决方案在50毫升的凝胶三次洗为30分钟。 </li><li>使用白色的光透射或扫描仪记录图像。另外，干凝胶可在一帧之间的玻璃纸张。 </li></ol> 3。临- Q翡翠检测糖化蛋白SDS - PAGE凝胶300 <ol><li> 2.1凝胶）的同时，负载在10-20％Tris -甘氨酸凝胶，其余样品。负载10＆万亩; ColorPlus预染蛋白Marker升。 </li><li>虽然凝胶是运行溶解DMF PRO - Q翡翠试剂和准备股票的修复，清洗和氧化性解决方案为300 PRO - Q翡翠染色以下产品与试剂盒提供的手册。 </li><li>电泳完成后，取出凝胶和从投它放置在一个塑料盒。 </li><li>修正加入100毫升的修复方案，并留在室温下轻轻摇动过夜的凝胶。 </li><li> 100毫升的洗涤液10至20分钟，在室温詹蒂莱鼓动清洗凝胶。重复用新鲜的洗涤液洗。 </li><li>轻轻摇动孵育30分钟，在氧化溶液25毫升凝胶氧化碳水化合物。 </li><li>步骤3.5中所述的凝胶洗净。 </li><li>凝胶洗涤准备的Pro - Q翡翠300试剂在步骤3.2至25 ml溶解的解决方案中加入500μL新鲜的PRO - Q 300翡翠染色染色试剂盒中提供的缓冲区。 </li><li>凝胶染色加入25毫升的步骤3.8准备污渍和潜伏在黑暗中轻轻摇动90至120分钟。 </li><li>重复步骤3.5中所描述的两个洗步骤。 </li><li>记录在300nm的紫外透射图像。使用80 kDa的标记标示，这是与临 - Q翡翠绿，紫外线图像重叠到白光图片显示预染阶梯的凝胶。 </li><li>比较Coomasie染色凝胶的Pro - Q翡翠染色凝胶步骤3.11 2.10步的图像。 </li></ol> 4。代表性的成果在蛋白质酶deglycosylation后迁移的变化如图2所示。比较对照样品（A组，泳道1）PNGase F治疗（去除N -糖链，2车道），和Deglycosylation混合（PNGase F，再加上远藤和exoglycosidases 取下 O -聚糖，车道3）。没有进一步减少（4车道）额外糖苷酶消化后的大小。除了大规模的变化，乐队变得更加清晰，为聚糖被删除。 A频段的17 kDa的标记（星号）下运行完全deglycosylatedhCGβ多肽（分子量：16 kDa）的代表。其他乐队可能来自不完全deglycosylation，或从多个身份不明的hCGβ样品中的蛋白质（见1巷）。 5日至7车道（对照组），显示相应的糖苷酶的乐队。 翡翠绿的糖蛋白染色显示面板B.此试剂氧化和污渍的一种蛋白质分子中存在的所有聚糖。因此信号强度下降hCGβ是酶deglycosylated（巷1号至4车道）。泳道3和4的剩余信号，表明聚糖的图案，所使用的酶有抗药性的存在。 4车道使用额外的糖苷酶，删除了一些额外的糖残基：蛋白质的迁移是相同的，但可以看出，在强度略有减少染色。抗糖基是目前在所有的蛋白质种类：翡翠绿（缺席的紫外线图像，括号内），说明他们广泛deglycosylated的某些频带均未检出。额外的数据支持hCGβ不均匀糖化的结论。低频段巷2号（箭头）是淡淡的翡翠的绿色形象，而2巷上带是光明的，表明许多聚糖组仍然存在。这些数据支持这样的结论，在小鼠细胞中表达的重组hCGβ包含多个 glycoforms 9。延长，而这些不同glycoforms由于一些多肽没有收到在每共识网站和/或一些聚糖聚糖的糖基化的内在异质性的人即使在相同的蛋白质不。 <img alt="图1" src="/files/ftp_upload/3749/3749fig1.jpg" /> 图1。典型的分泌或细胞表面的糖蛋白糖基化模式。 <img alt="图2" src="/files/ftp_upload/3749/3749fig2.jpg" /> 图2 SDS - PAGE凝胶酶deglycosylationhCGβ。 A组显示一个Coomasie蓝染色，而B组临- Q翡翠300糖化蛋白的可视化结果。样品编号：1，hCGβ控制; 2，PNGase F消化; 3，Deglycosylation混合消化; 4，Deglycosylation Mix Plus的exoglycosidases消化，样品5至7试剂控制（O型，O型糖苷酶甘油; GNA，β- N Acetylglucosaminidase;加仑/ FUC，β1- 3半乳糖苷酶，α1- 3，6半乳糖苷酶和α1- 2岩藻糖苷酶; GalNA，α- N - Acetylglalactosaminidase;神经，神经氨酸酶; PNGase PNGase F）

<ol>
	<li>Spiro, R. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+glycosylation:+nature,+distribution,+enzymatic+formation,+and+disease+implications+of+glycopeptide+bonds.">Protein glycosylation: nature, distribution, enzymatic formation, and disease implications of glycopeptide bonds.</a> Glycobiology. 12 (4), 43-56 (2002).</li><li>Ohtsubo, K., Marth, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glycosylation+in+Cellular+Mechanisms+of+Health+and+Disease.">Glycosylation in Cellular Mechanisms of Health and Disease.</a> Cell. 126, 855-867 (2006).</li><li>Arnold, J. N., Wormald, M. R., Sim, R. B., Rudd, P. M., Dwek, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+impact+of+glycosylation+on+the+biological+function+and+structure+of+human+immunoglobulins.">The impact of glycosylation on the biological function and structure of human immunoglobulins.</a> Annual Review of Immunology. 25, 21-50 (2007).</li><li>Mitra, N., Sinha, S., Ramya, T. N. C., Surolia, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=N-linked+oligosaccharides+as+outfitters+for+glycoprotein+folding,+form+and+function.">N-linked oligosaccharides as outfitters for glycoprotein folding, form and function.</a> Trends in Biochemical Sciences. 31, 156-163 (2006).</li><li>Carlsen, R. B., Bahl, O. P., Swaminathan, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+chorionic+gonadotropin.+Linear+amino+acid+sequence+of+the+beta+subunit.">Human chorionic gonadotropin. Linear amino acid sequence of the beta subunit.</a> Journal of Biological Chemistry. 248 (19), 6810-6812 (1973).</li><li>Tarentino, A. L., Plummer, T. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Deglycosylation+of+Asparagine-linked+Glycans+by+PNGaseF.">Deglycosylation of Asparagine-linked Glycans by PNGaseF.</a> Trends in Glycoscience and Glycotechnology. 5 (23), 163-170 (1993).</li><li>Tretter, V., Altmann, F., Marz, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Peptide-N4-(N-acetyl-&#946;-glucosaminyl)asparagine+amidase+F+cannot+release+glycans+with+fucose+attached+&#945;1-3+to+the+asparagine-linked+N-acetylglucosamine+residue.">Peptide-N4-(N-acetyl-&#946;-glucosaminyl)asparagine amidase F cannot release glycans with fucose attached &#945;1-3 to the asparagine-linked N-acetylglucosamine residue.</a> European Journal of Biochemistry. 199, 647-652 (1991).</li><li>Koutsioulis, D., Landry, D., Guthrie, 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=Novel+endo-&#945;-N-acetylgalactosaminidases+with+broader+substrate+specificity.">Novel endo-&#945;-N-acetylgalactosaminidases with broader substrate specificity.</a> Glycobiology. 18 (10), 799-805 (2008).</li><li>Thakur, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Profiling+the+glycoforms+of+the+intact+alpha+subunit+of+recombinant+human+chorionic+gonadotropin+by+high-resolution+capillary+electrophoresis-mass+spectrometry.">Profiling the glycoforms of the intact alpha subunit of recombinant human chorionic gonadotropin by high-resolution capillary electrophoresis-mass spectrometry.</a> Analytical Chemistry. 81 (2), 8900-8907 (2009).</li><li>Park, S., Lee, M. R., Shin, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Carbohydrate+microarrays+as+powerful+tools+in+studies+of+carbohydrate-mediated+biological+processes.">Carbohydrate microarrays as powerful tools in studies of carbohydrate-mediated biological processes.</a> Chemical Communications. 37, 4389-4399 (2008).</li><li>Zachara, N. E., Vosseller, K., Hart, G. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+and+analysis+of+proteins+modified+by+O-linked+N-acetylglucosamine.">Detection and analysis of proteins modified by O-linked N-acetylglucosamine.</a> Current Protocols in Molecular Biology. 95, 17-17 (2011).</li><li>Varki, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Essentials of Glycobiology. , (2008).</li></ol>

作者受雇于New England Biolabs公司，从而产生许多本文中使用的试剂。

这里介绍的方法使用酶deglycosylation和SDS - PAGE可以提供有价值的信息有关的蛋白糖基化状态，而聚糖特定试剂方便对数据的解释。此协议的目的是为蛋白质糖基化的初步研究，它特别适用于哺乳动物细胞分泌和膜糖蛋白：在这种情况下选择的酶将专门删除所有的 N -糖链， 或N -糖链加上最常见的糖扩大和形成的 O -糖链的核心。糖苷酶具有额外的优势正在温和，用化学deglycosylation方法相比，保持糖和蛋白质的骨干的完整性。 为了阐明入住率（氨基酸是糖化），糖基化的程度，或确定的精细结构的多聚糖，更先进的技术，如米屁股光谱仪，液相色谱仪或核磁共振是必需的。 由于其简单，在这个协议中的几个步骤可以进行调整，替换，和/或合并，以适应不同的实验需要。然而，为了获得结果，可以清楚地解释重要的是要了解它的长处和局限性。首先，特异性和糖苷酶的纯度是至关重要的：只有良好的特点测试蛋白酶和其他污染活动的酶，应使用。不幸的是，没有标准的单位定义为糖苷酶，根据制造商的规格，用户应确定适当的替代。二，谨慎选择的检测是必要的：A）蛋白染色试剂是有用的，只有在一个相对分子质量的重大转变的deglycosylation结果。并不总是得到这样一个明确的结果如下所示。在其他情况下，我们所看到的异常MIdeglycosylation后格拉逊（远远低于预测的分子量，或更慢迁移） 。这种现象是没有很好地理解，但也可以说， 任何在迁移变化是证据表明，该蛋白已deglycosylated B）糖与抗体检测呈现独特的挑战限制其适用性。它已经很难产生一般反聚糖抗体，它们通常是对一个复杂的糖链的目标，这限制了它们的使用提出。此外，一些单克隆抗聚糖抗体显示不受欢迎的交叉反应 10。彗星）凝集素（糖亲和力内在蛋白）是非常适合对糖的检测，加上他们给聚糖结构上的洞察力。然而，并非所有这些有一个狭窄的特异性，很多都只是部分的特点（这意味着他们可能有未知的亲和力）。因此积极的外源凝集素染色提供指示，而不是地理标志的存在证明，VEN糖D）化学标记试剂盒（基于碘酸氧化糖）的首选方法染色所有的糖蛋白，因此非常适合跟随deglycosylation。 处理未知样品时，这是一个很好的做法，包括糖蛋白控制。胎球蛋白是一个现成的N - 和 O -糖蛋白，绒毛膜促性腺激素（两个亚基）也是一个不错的选择。牛血清白蛋白（BSA）可以用来作为阴性对照。但是，应该指出，一些非糖基化蛋白反应轻微的Pro - Q 300翡翠，特别是在高浓度时使用。非糖基化的分子量标准，如在本议定书中使用的预染蛋白质标记，显示锐利带的优势。不仅是这些蛋白质（80 kDa）的反应到临 - Q翡翠300之一的机会。因此，用户可能要运行的一种糖蛋白标准的阶梯，而不是，如Invitroge糖甘蔗N. 最后，简单的凝胶nucleocytosolic糖蛋白（这是一个单一的O型葡萄糖修改）的检测是可能的使用单克隆抗体，沿β- N - Acetylglucosaminidase 特异性控制11。超越了这项技术的描述是这篇文章的范围，但它应该提到糖苷酶在细胞中存在的许多其他糖蛋白和糖复合物的研究是非常有用的工具。一个所有已知的糖基化形式的综合治疗，可以发现在第二版的“糖生物学基础”，可在NCBI的书柜（书架编号：;：20301239关键词NBK1908）免费在线12。

<table border="1"><tbody><tr><td> 试剂名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论 </td></tr><tr><td>人绒毛膜促性腺激素β </td><td>西格玛爱秩序</td><td> C6572 </td><td></td></tr><tr><td> PCR管</td><td> VWR </td><td> 20170-004 </td><td></td></tr><tr><td> PCR扩增热循环仪</td><td>应用生物系统公司</td><td> 4359659 </td><td></td></tr><tr><td> PNGase F </td><td> New England Biolabs公司</td><td> P0704 </td><td>提供与缓冲区</td></tr><tr><td>蛋白质Deglycosylation组合</td><td> New England Biolabs公司</td><td> P6039 </td><td>提供与缓冲区</td></tr><tr><td> α- N - Acetylglalactosaminidase </td><td> New England Biolabs公司</td><td> P0734 </td><td>提供带缓冲</td></tr><tr><td> α1- 2岩藻糖苷酶</td><td> New England Biolabs公司</td><td> P0724 </td><td>提供带缓冲</td></tr><tr><td> α1- 3，6半乳糖苷酶</td><td> New England Biolabs公司</td><td> P0731 </td><td>提供带缓冲</td></tr><tr><td> β1- 3半乳糖苷酶</td><td> New England Biolabs公司</td><td> P0726 </td><td>提供带缓冲</td></tr><tr><td> 10X缓冲G7（500 mM磷酸钠） </td><td> New England Biolabs公司</td><td> --- </td><td>提供与PNGaseF或Degl混合</td></tr><tr><td> 10X糖蛋白变性缓冲液（5％SDS，400毫米数码地面电视） </td><td> New England Biolabs公司</td><td> --- </td><td>提供与PNGaseF或Degl混合</td></tr><tr><td> 10％NP - 40 </td><td> New England Biolabs公司</td><td> --- </td><td>提供与PNGaseF或Degl混合</td></tr><tr><td> 3X SDS上样缓冲液（187.5毫米的Tris - HCl pH值6.8，6％SDS，30％甘油，0.03％溴ophenol蓝色） </td><td> New England Biolabs公司</td><td> B7703 </td><td>提供与1.25M数码地面电视</td></tr><tr><td> ColorPlus预染蛋白Marker </td><td> New England Biolabs公司</td><td> P7709 </td><td></td></tr><tr><td> 10-20％的Tris -甘氨酸Multigel </td><td>丽悦生物有限公司</td><td> DCB - 414893 </td><td></td></tr><tr><td>盒式电​​泳单元</td><td>丽悦生物有限公司</td><td> DCB - 303111 </td><td></td></tr><tr><td>电泳电源的EPS 301 </td><td> GE医疗集团</td><td> 18-1130-01 </td><td></td></tr><tr><td>临- Q翡翠300糖蛋白染色试剂盒</td><td> Invitrogen公司</td><td> P - 21857 </td><td></td></tr><tr><td>艳蓝R </td><td>西格玛爱秩序</td><td> B0149 </td><td></td></tr><tr><td> AlphaImager惠普系统</td><td>细胞生物科技公司</td><td> 92-13823-00 </td><td></td></tr></tbody></table>

identification and characterization of protein glycosylation using specific endo- and exoglycosidases

此外共价连接的糖，糖基化，是一个重要的翻译后修饰的蛋白质，可以显着影响，如细 ​​胞粘附分子贩运，清关，和信号转导1-4的过程。在真核生物中，在分泌途径中最常见的糖基化修改，增加共识，天门冬素剩余物（N -连接）;或在丝氨酸或苏氨酸残基（O型连锁）（图1 ）。在真核生物中是高度保守的N -聚糖合成的起始，而最终产品可以在不同物种间，组织，或蛋白质大大不同。有些聚糖保持未修改“（”高甘露糖N -糖链“），或在高尔基（” 复杂的N -糖链“）进一步处理。更大的多样性是O型聚糖，开始与一个共同的 N -乙酰半乳糖胺（半乳糖胺）残留在动物细胞中，但不同的低等生物 1 。 耳鼻喉科“&gt;蛋白质的糖基化的详细分析本身就是一个领域，需要丰富的资源和专业知识，正确执行。然而，各种可用的酶，删除糖（糖苷）成为可能的糖基化的现状有一个总体思路在一个标准的实验室环境中的蛋白质，在这里我们说明分析模型糖蛋白糖苷酶的使用。重组人绒毛膜促性腺激素β（hCGβ）， 它带有两个N -糖链和四个O型聚糖5，该技术只需要简单仪器和典型的消耗品，它可以很容易地适应多种糖蛋白样品的分析。 几种酶可用于并行研究的一种糖蛋白。 PNGase F是能够去除几乎所有类型的N -连接聚糖6,7。 为 O -糖链，有没有可用的酶可以切割完整的低聚糖从日E蛋白的骨干。相反，O型聚糖修剪exoglycosidases一个短的核心，是那么容易由 O -糖苷酶删除。蛋白质Deglycosylation结构包含PNGase F，O -糖苷酶，神经氨酸酶（唾液酸），β1- 4半乳糖苷酶和β- N - Acetylglucosaminidase。它是用来同时去除N -糖链和一些O型聚糖8。最后，Deglycosylation混合补充的其他exoglycosidases混合物（α- N - Acetylgalactosaminidase，α1- 2岩藻糖苷酶，α1- 3，6半乳糖苷酶，β1- 3半乳糖苷酶），这有助于消除否则抗单糖，可目前在某些O -聚糖。 SDS-PAGE/Coomasie蓝色是用于可视化蛋白质迁移糖苷酶治疗前后的差异。此外，糖特定的染色方法，ProQ翡翠- 300，显示减少信号聚糖是帅客ssively删除。此协议是专为少量的糖蛋白（0.5％到2微克）的分析，虽然酶deglycosylation可以扩大规模，以适应需要较大的蛋白质数量。

Watch this Scientific Journal Video about Identification and Characterization of Protein Glycosylation using Specific Endo- and Exoglycosidases at JoVE.com

Identification and Characterization of Protein Glycosylation using Specific Endo- and Exoglycosidases

使用特定的糖苷酶，以消除通过SDS - PAGE的糖蛋白的糖是一种有价值的的方法来检测蛋白质样品聚糖修改初始糖生物学研究的是一个不错的选择。以下deglycosylation变化可以检测凝胶的流动性的变化或与多糖敏感的试剂染色。

使用具体远藤和exoglycosidases糖基化蛋白质的鉴定和表征

Glycosylation, the addition of covalently linked sugars, is a major post-translational modification of proteins that can significantly affect processes ...

identification-characterization-protein-glycosylation-using-specific

Research

JoVE Journal

Biology

36.4K 次观看.  New England Biolabs. 使用特定的糖苷酶，以消除通过SDS - PAGE的糖蛋白的糖是一种有价值的的方法来检测蛋白质样品聚糖修改初始糖生物学研究的是一个不错的选择。以下deglycosylation变化可以检测凝胶的流动性的变化或与多糖敏感的试剂染色。

视频: Identification and Characterization of Protein Glycosylation using Specific Endo- and Exoglycosidases

Identification of protein complexes with quantitative proteomics in S. cerevisiae

Lipids are the building blocks of cellular membranes that function as barriers and in compartmentalization of cellular processes, and recently, as important intracellular signalling molecules. However, unlike proteins, lipids are small hydrophobic molecules that traffic primarily by poorly described nonvesicular routes, which are hypothesized to occur at membrane contact sites (MCSs). MCSs are regions where the endoplasmic reticulum (ER) makes direct physical contact with a partnering organelle, e.g., plasma membrane (PM). The ER portion of ER-PM MCSs is enriched in lipid-synthesizing enzymes, suggesting that lipid synthesis is directed to these sites and implying that MCSs are important for lipid traffic. Yeast is an ideal model to study ER-PM MCSs because of their abundance, with over 1000 contacts per cell, and their conserved nature in all eukaryotes. Uncovering the proteins that constitute MCSs is critical to understanding how lipids traffic is accomplished in cells, and how they act as signaling molecules. We have found that an ER called Scs2p localize to ER-PM MCSs and is important for their formation. We are focused on uncovering the molecular partners of Scs2p. Identification of protein complexes traditionally relies on first resolving purified protein samples by gel electrophoresis, followed by in-gel digestion of protein bands and analysis of peptides by mass spectrometry. This often limits the study to a small subset of proteins. Also, protein complexes are exposed to denaturing or non-physiological conditions during the procedure. To circumvent these problems, we have implemented a large-scale quantitative proteomics technique to extract unbiased and quantified data. We use stable isotope labeling with amino acids in cell culture (SILAC) to incorporate staple isotope nuclei in proteins in an untagged control strain. Equal volumes of tagged culture and untagged, SILAC-labeled culture are mixed together and lysed by grinding in liquid nitrogen. We then carry out an affinity purification procedure to pull down protein complexes. Finally, we precipitate the protein sample, which is ready for analysis by high-performance liquid chromatography/ tandem mass spectrometry. Most importantly, proteins in the control strain are labeled by the heavy isotope and will produce a mass/ charge shift that can be quantified against the unlabeled proteins in the bait strain. Therefore, contaminants, or unspecific binding can be easily eliminated. By using this approach, we have identified several novel proteins that localize to ER-PM MCSs. Here we present a detailed description of our approach. 

Here we describe a new quantitative proteomics technique for identifying protein complexes in Saccharomyces cerevisiae. In this study, we have used the SILAC method together with affinity purification followed by tandem mass spectrometry to identify with high specificity the binding partners of an ER protein, Scs2p.

鉴定与定量蛋白质组学在酿酒酵母中的蛋白质复合物

Lipids are the building blocks of cellular membranes that function as barriers and in compartmentalization of cellular processes, and recently, as ...

科研

生物学

Using the GELFREE 8100 Fractionation System for Molecular Weight-Based Fractionation with Liquid Phase Recovery

The GELFREE 8100 Fractionation System is a novel protein fractionation system designed to maximize protein recovery during molecular weight based fractionation. The system is comprised of single-use, 8-sample capacity cartridges and a benchtop GELFREE Fractionation Instrument. During separation, a constant voltage is applied between the anode and cathode reservoirs, and each protein mixture is electrophoretically driven from a loading chamber into a specially designed gel column gel. Proteins are concentrated into a tight band in a stacking gel, and separated based on their respective electrophoretic mobilities in a resolving gel. As proteins elute from the column, they are trapped and concentrated in liquid phase in the collection chamber, free of the gel. The instrument is then paused at specific time intervals, and fractions are collected using a pipette. This process is repeated until all desired fractions have been collected. If fewer than 8 samples are run on a cartridge, any unused chambers can be used in subsequent separations.
This novel technology facilitates the quick and simple separation of up to 8 complex protein mixtures simultaneously, and offers several advantages when compared to previously available fractionation methods. This system is capable of fractionating up to 1mg of total protein per channel, for a total of 8mg per cartridge. Intact proteins over a broad mass range are separated on the basis of molecular weight, retaining important physiochemical properties of the analyte. The liquid phase entrapment provides for high recovery while eliminating the need for band or spot cutting, making the fractionation process highly reproducible1.

The accompanying video describes the use of the GELFREE 8100 Fractionation System, which partitions complex protein samples on the basis of molecular weight and recovers the fractions in liquid phase. The video describes how the technology works, how it is used, and provides resultant data, with polyacrylamide gel electrophoresis analysis of fractionated bovine liver homogenate.

液相恢复GELFREE 8100分馏系统使用基于分子量分馏

The GELFREE 8100 Fractionation System is a novel protein fractionation system designed to maximize protein recovery during molecular weight based ...

Identification of Protein Interacting Partners Using Tandem Affinity Purification

A critical and often limiting step in understanding the function of host and viral proteins is the identification of interacting cellular or viral protein partners. There are many approaches that allow the identification of interacting partners, including the yeast two hybrid system, as well as pull down assays using recombinant proteins and immunoprecipitation of endogenous proteins followed by mass spectrometry identification1. Recent studies have highlighted the utility of double-affinity tag mediated purification, coupled with two specific elution steps in the identification of interacting proteins. This approach, termed Tandem Affinity Purification (TAP), was initially used in yeast2,3 but more recently has been adapted to use in mammalian cells4-8.
As proof-of-concept we have established a tandem affinity purification (TAP) method using the well-characterized eukaryotic translation initiation factor eIF4E9,10.The cellular translation factor eIF4E is a critical component of the cellular eIF4F complex involved in cap-dependent translation initiation10. The TAP tag used in the current study is composed of two Protein G units and a streptavidin binding peptide separated by a Tobacco Etch Virus (TEV) protease cleavage sequence. The TAP tag used in the current study is composed of two Protein G units and a streptavidin binding peptide separated by a Tobacco Etch Virus (TEV) protease cleavage sequence8. To forgo the need for the generation of clonal cell lines, we developed a rapid system that relies on the expression of the TAP-tagged bait protein from an episomally maintained plasmid based on pMEP4 (Invitrogen). Expression of tagged murine eIF4E from this plasmid was controlled using the cadmium chloride inducible metallothionein promoter.
Lysis of the expressing cells and subsequent affinity purification via binding to rabbit IgG agarose, TEV protease cleavage, binding to streptavidin linked agarose and subsequent biotin elution identified numerous proteins apparently specific to the eIF4E pull-down (when compared to control cell lines expressing the TAP tag alone). The identities of the proteins were obtained by excision of the bands from 1D SDS-PAGE and subsequent tandem mass spectrometry. The identified components included the known eIF4E binding proteins eIF4G and 4EBP-1. In addition, other components of the eIF4F complex, of which eIF4E is a component were identified, namely eIF4A and Poly-A binding protein. The ability to identify not only known direct binding partners as well as secondary interacting proteins, further highlights the utility of this approach in the characterization of proteins of unknown function.

Tandem affinity purification is a robust approach for the identification of protein binding partners. As proof of concept, this methodology was applied to the well-characterized translation initiation factor eIF4E to co-precipitate the host cell factors involved in translation initiation. This method is easily adapted to any cellular or viral protein.

鉴定蛋白质相互作用的合作伙伴使用串联亲和纯化

A critical and often limiting step in understanding the function of host and viral proteins is the identification of interacting cellular or viral protein ...

Chemically-blocked Antibody Microarray for Multiplexed High-throughput Profiling of Specific Protein Glycosylation in Complex Samples

In this study, we describe an effective protocol for use in a multiplexed high-throughput antibody microarray with glycan binding protein detection that allows for the glycosylation profiling of specific proteins. Glycosylation of proteins is the most prevalent post-translational modification found on proteins, and leads diversified modifications of the physical, chemical, and biological properties of proteins. Because the glycosylation machinery is particularly susceptible to disease progression and malignant transformation, aberrant glycosylation has been recognized as early detection biomarkers for cancer and other diseases. However, current methods to study protein glycosylation typically are too complicated or expensive for use in most normal laboratory or clinical settings and a more practical method to study protein glycosylation is needed. The new protocol described in this study makes use of a chemically blocked antibody microarray with glycan-binding protein (GBP) detection and significantly reduces the time, cost, and lab equipment requirements needed to study protein glycosylation. In this method, multiple immobilized glycoprotein-specific antibodies are printed directly onto the microarray slides and the N-glycans on the antibodies are blocked. The blocked, immobilized glycoprotein-specific antibodies are able to capture and isolate glycoproteins from a complex sample that is applied directly onto the microarray slides. Glycan detection then can be performed by the application of biotinylated lectins and other GBPs to the microarray slide, while binding levels can be determined using Dylight 549-Streptavidin. Through the use of an antibody panel and probing with multiple biotinylated lectins, this method allows for an effective glycosylation profile of the different proteins found in a given human or animal sample to be developed.
Introduction
Glycosylation of protein, which is the most ubiquitous post-translational modification on proteins, modifies the physical, chemical, and biological properties of a protein, and plays a fundamental role in various biological processes1-6. Because the glycosylation machinery is particularly susceptible to disease progression and malignant transformation, aberrant glycosylation has been recognized as early detection biomarkers for cancer and other diseases 7-12. In fact, most current cancer biomarkers, such as the L3 fraction of &alpha;-1 fetoprotein (AFP) for hepatocellular carcinoma 13-15, and CA199 for pancreatic cancer 16, 17 are all aberrant glycan moieties on glycoproteins. However, methods to study protein glycosylation have been complicated, and not suitable for routine laboratory and clinical settings. Chen et al. has recently invented a chemically blocked antibody microarray with a glycan-binding protein (GBP) detection method for high-throughput and multiplexed profile glycosylation of native glycoproteins in a complex sample 18. In this affinity based microarray method, multiple immobilized glycoprotein-specific antibodies capture and isolate glycoproteins from the complex mixture directly on the microarray slide, and the glycans on each individual captured protein are measured by GBPs. Because all normal antibodies contain N-glycans which could be recognized by most GBPs, the critical step of this method is to chemically block the glycans on the antibodies from binding to GBP. In the procedure, the cis-diol groups of the glycans on the antibodies were first oxidized to aldehyde groups by using NaIO4 in sodium acetate buffer avoiding light. The aldehyde groups were then conjugated to the hydrazide group of a cross-linker, 4-(4-N-MaleimidoPhenyl)butyric acid Hydrazide HCl (MPBH), followed by the conjugation of a dipeptide, Cys-Gly, to the maleimide group of the MPBH. Thus, the cis-diol groups on glycans of antibodies were converted into bulky none hydroxyl groups, which hindered the lectins and other GBPs bindings to the capture antibodies. This blocking procedure makes the GBPs and lectins bind only to the glycans of captured proteins. After this chemically blocking, serum samples were incubated with the antibody microarray, followed by the glycans detection by using different biotinylated lectins and GBPs, and visualized with Cy3-streptavidin. The parallel use of an antibody panel and multiple lectin probing provides discrete glycosylation profiles of multiple proteins in a given sample 18-20. This method has been used successfully in multiple different labs 1, 7, 13, 19-31. However, stability of MPBH and Cys-Gly, complicated and extended procedure in this method affect the reproducibility, effectiveness and efficiency of the method. In this new protocol, we replaced both MPBH and Cys-Gly with one much more stable reagent glutamic acid hydrazide (Glu-hydrazide), which significantly improved the reproducibility of the method, simplified and shorten the whole procedure so that the it can be completed within one working day. In this new protocol, we describe the detailed procedure of the protocol which can be readily adopted by normal labs for routine protein glycosylation study and techniques which are necessary to obtain reproducible and repeatable results.

In this study, we describe an improved protocol for a multiplexed high-throughput antibody microarray with lectin detection method that can be used in glycosylation profiling of specific proteins. This protocol features new reliable reagents and significantly reduces the time, cost, and lab equipment requirements as compared to the previous procedure.

化学阻断抗体芯片复用高通量的复杂样品中特定蛋白糖基化的剖析

 In this study, we describe an effective protocol for use in a multiplexed high-throughput antibody microarray with glycan binding protein detection that ...

Method for the Isolation and Identification of mRNAs, microRNAs and Protein Components of Ribonucleoprotein Complexes from Cell Extracts using RIP-Chip

As a result of the development of high-throughput sequencing and efficient microarray analysis, global gene expression analysis has become an easy and readily available form of data collection. In many research and disease models however, steady state levels of target gene mRNA does not always directly correlate with steady state protein levels. Post-transcriptional gene regulation is a likely explanation of the divergence between the two. Driven by the binding of RNA Binding Proteins (RBP), post-transcriptional regulation affects mRNA localization, stability and translation by forming a Ribonucleoprotein (RNP) complex with target mRNAs. Identifying these unknown de novo mRNA targets from cellular extracts in the RNP complex is pivotal to understanding mechanisms and functions of the RBP and their resulting effect on protein output. This protocol outlines a method termed RNP immunoprecipitation-microarray (RIP-Chip), which allows for the identification of specific mRNAs associated in the ribonucleoprotein complex, under changing experimental conditions, along with options to further optimize an experiment for the individual researcher. With this important experimental tool, researchers can explore the intricate mechanisms associated with post-transcriptional gene regulation as well as other ribonucleoprotein interactions.

A step by step protocol to isolating and identifying RNA associated complexes through RIP-Chip.

使用RIP-芯片的核糖核蛋白复合物的基因，小分子RNA和蛋白质组分的分离与鉴定细胞提取物的方法

As a result of the development of high-throughput sequencing and efficient microarray analysis, global gene expression analysis has become an easy and ...

Identification of Protein Complexes in Escherichia coli using Sequential Peptide Affinity Purification in Combination with Tandem Mass Spectrometry

Since most cellular processes are mediated by macromolecular assemblies, the systematic identification of protein-protein interactions (PPI) and the identification of the subunit composition of multi-protein complexes can provide insight into gene function and enhance understanding of biological systems1, 2. Physical interactions can be mapped with high confidence vialarge-scale isolation and characterization of endogenous protein complexes under near-physiological conditions based on affinity purification of chromosomally-tagged proteins in combination with mass spectrometry (APMS). This approach has been successfully applied in evolutionarily diverse organisms, including yeast, flies, worms, mammalian cells, and bacteria1-6. In particular, we have generated a carboxy-terminal Sequential Peptide Affinity (SPA) dual tagging system for affinity-purifying native protein complexes from cultured gram-negative Escherichia coli, using genetically-tractable host laboratory strains that are well-suited for genome-wide investigations of the fundamental biology and conserved processes of prokaryotes1, 2, 7. Our SPA-tagging system is analogous to the tandem affinity purification method developed originally for yeast8, 9, and consists of a calmodulin binding peptide (CBP) followed by the cleavage site for the highly specific tobacco etch virus (TEV) protease and three copies of the FLAG epitope (3X FLAG), allowing for two consecutive rounds of affinity enrichment. After cassette amplification, sequence-specific linear PCR products encoding the SPA-tag and a selectable marker are integrated and expressed in frame as carboxy-terminal fusions in a DY330 background that is induced to transiently express a highly efficient heterologous bacteriophage lambda recombination system10. Subsequent dual-step purification using calmodulin and anti-FLAG affinity beads enables the highly selective and efficient recovery of even low abundance protein complexes from large-scale cultures. Tandem mass spectrometry is then used to identify the stably co-purifying proteins with high sensitivity (low nanogram detection limits).
Here, we describe detailed step-by-step procedures we commonly use for systematic protein tagging, purification and mass spectrometry-based analysis of soluble protein complexes from E. coli, which can be scaled up and potentially tailored to other bacterial species, including certain opportunistic pathogens that are amenable to recombineering. The resulting physical interactions can often reveal interesting unexpected components and connections suggesting novel mechanistic links. Integration of the PPI data with alternate molecular association data such as genetic (gene-gene) interactions and genomic-context (GC) predictions can facilitate elucidation of the global molecular organization of multi-protein complexes within biological pathways. The networks generated for E. coli can be used to gain insight into the functional architecture of orthologous gene products in other microbes for which functional annotations are currently lacking.

Identification of Protein Complexes in Escherichia coli using Sequential Peptide Affinity Purification in Combination with Tandem Mass Spectrometry

Affinity purification of tagged proteins in combination with mass spectrometry (APMS) is a powerful method for the systematic mapping of protein interaction networks and for investigating the mechanistic basis of biological processes. Here, we describe an optimized sequential peptide affinity (SPA) APMS procedure developed for the bacterium Escherichia coli that can be used to isolate and characterize stable multi-protein complexes to near homogeneity even starting from low copy numbers per cell.

蛋白复合物的鉴定<em&gt;大肠杆菌</em使用顺序肽亲和纯化技术相结合串联质谱法

Since most cellular processes are mediated by macromolecular assemblies, the systematic identification of protein-protein interactions (PPI) and the ...

Generation, Purification, and Characterization of Cell-invasive DISC1 Protein Species

Protein aggregation is seen as a general hallmark of chronic, degenerative brain conditions like, for example, in the neurodegenerative diseases Alzheimer's disease (A&beta;, tau), Parkinson's Disease (&alpha;-synuclein), Huntington's disease (polyglutamine, huntingtin), and others. Protein aggregation is thought to occur due to disturbed proteostasis, i.e. the imbalance between the arising and degradation of misfolded proteins. Of note, the same proteins are found aggregated in sporadic forms of these diseases that are mutant in rare variants of familial forms.
Schizophrenia is a chronic progressive brain condition that in many cases goes along with a permanent and irreversible cognitive deficit. In a candidate gene approach, we investigated whether Disrupted-in-schizophrenia 1 (DISC1), a gene cloned in a Scottish family with linkage to chronic mental disease1, 2, could be found as insoluble aggregates in the brain of sporadic cases of schizophrenia3. Using the SMRI CC, we identified in approximately 20 % of cases with CMD but not normal controls or patients with neurodegenerative diseases sarkosyl-insoluble DISC1 immunoreactivity after biochemical fractionation. Subsequent studies in vitro revealed that the aggregation propensity of DISC1 was influenced by disease-associated polymorphism S704C4, and that DISC1 aggresomes generated in vitro were cell-invasive5, similar to what had been shown for A&beta;6, tau7-9, &alpha;-synuclein10, polyglutamine11, or SOD1 aggregates12. These findings prompted us to propose that at least a subset of cases with CMD, those with aggregated DISC1 might be protein conformational disorders.
 Here we describe how we generate DISC1 aggresomes in mammalian cells, purify them on a sucrose gradient and use them for cell-invasiveness studies. Similarly, we describe how we generate an exclusively multimeric C-terminal DISC1 fragment, label and purify it for cell invasiveness studies. Using the recombinant multimers of DISC1 we achieve similar cell invasiveness as for a similarly labeled synthetic &alpha;-synuclein fragment. We also show that this fragment is taken up in vivo when stereotactically injected into the brain of recipient animals.

The generation, purification and cell invasion of intracellular, cytoplasmic full length DISC1 protein aggresomes from cell cultures and of a labeled, multimeric recombinant DISC1 protein fragment in E. coli are described. Cell invasiveness is shown for recipient cells in cell culture and for neurons in vivo after stereotactical brain inoculation.

侵入细胞DISC1蛋白种类的产生，纯化及表征

Protein aggregation is seen as a general hallmark of chronic, degenerative brain conditions like, for example, in the neurodegenerative diseases ...

Pyrosequencing for Microbial Identification and Characterization

Pyrosequencing is a versatile technique that facilitates microbial genome sequencing that can be used to identify bacterial species, discriminate bacterial strains and detect genetic mutations that confer resistance to anti-microbial agents. The advantages of pyrosequencing for microbiology applications include rapid and reliable high-throughput screening and accurate identification of microbes and microbial genome mutations. Pyrosequencing involves sequencing of DNA by synthesizing the complementary strand a single base at a time, while determining the specific nucleotide being incorporated during the synthesis reaction. The reaction occurs on immobilized single stranded template DNA where the four deoxyribonucleotides (dNTP) are added sequentially and the unincorporated dNTPs are enzymatically degraded before addition of the next dNTP to the synthesis reaction. Detection of the specific base incorporated into the template is monitored by generation of chemiluminescent signals. The order of dNTPs that produce the chemiluminescent signals determines the DNA sequence of the template. The real-time sequencing capability of pyrosequencing technology enables rapid microbial identification in a single assay. In addition, the pyrosequencing instrument, can analyze the full genetic diversity of anti-microbial drug resistance, including typing of SNPs, point mutations, insertions, and deletions, as well as quantification of multiple gene copies that may occur in some anti-microbial resistance patterns.

Pyrosequencing is a versatile technique that facilitates microbial genome sequencing that can be used to identify bacterial species, discriminate bacterial strains, and detect genetic mutations that confer resistance to anti-microbial agents. In this video, the procedure for microbial amplicon generation, amplicon pyrosequencing, and DNA sequence analysis will be demonstrated.

焦磷酸测序的微生物鉴定和表征

Pyrosequencing is a versatile technique that facilitates microbial genome sequencing that can be used to identify bacterial species, discriminate ...

Isolation of Specific Genomic Regions and Identification of Associated Molecules by enChIP

The identification of molecules associated with specific genomic regions of interest is required to understand the mechanisms of regulation of the functions of these regions. To enable the non-biased identification of molecules interacting with a specific genomic region of interest, we recently developed the engineered DNA-binding molecule-mediated chromatin immunoprecipitation (enChIP) technique. Here, we describe how to use enChIP to isolate specific genomic regions and identify the associated proteins and RNAs. First, a genomic region of interest is tagged with a transcription activator-like (TAL) protein or a clustered regularly interspaced short palindromic repeats (CRISPR) complex consisting of a catalytically inactive form of Cas9 and a guide RNA. Subsequently, the chromatin is crosslinked and fragmented by sonication. The tagged locus is then immunoprecipitated and the crosslinking is reversed. Finally, the proteins or RNAs that are associated with the isolated chromatin are subjected to mass spectrometric or RNA sequencing analyses, respectively. This approach allows the successful identification of proteins and RNAs associated with a genomic region of interest.

The identification of molecules associated with specific genomic regions of interest is required to understand the mechanisms of regulation of the functions of these regions. This protocol describes procedures to perform engineered DNA-binding molecule-mediated chromatin imunoprecipitation (enChIP) for identification of proteins and RNAs associated with a specific genomic region.

具体的基因组区域及相关分子鉴定enChIP隔离

The identification of molecules associated with specific genomic regions of interest is required to understand the mechanisms of regulation of the ...

Analysis of SCAP N-glycosylation and Trafficking in Human Cells

Elevated lipogenesis is a common characteristic of cancer and metabolic diseases. Sterol regulatory element-binding proteins (SREBPs), a family of membrane-bound transcription factors controlling the expression of genes important for the synthesis of cholesterol, fatty acids and phospholipids, are frequently upregulated in these diseases. In the process of SREBP nuclear translocation, SREBP-cleavage activating protein (SCAP) plays a central role in the trafficking of SREBP from the endoplasmic reticulum (ER) to the Golgi and in subsequent proteolysis activation. Recently, we uncovered that glucose-mediated N-glycosylation of SCAP is a prerequisite condition for the exit of SCAP/SREBP from the ER and movement to the Golgi. N-glycosylation stabilizes SCAP and directs SCAP/SREBP trafficking. Here, we describe a protocol for the isolation of membrane fractions in human cells and for the preparation of the samples for the detection of SCAP N-glycosylation and total protein by using western blot. We further provide a method to monitor SCAP trafficking by using confocal microscopy. This protocol is appropriate for the investigation of SCAP N-glycosylation and trafficking in mammalian cells.

Analysis of SCAP N-glycosylation and Trafficking in Human Cells

We describe a modified method for membrane fraction isolation from human cells and sample preparation for the detection of SCAP N-glycosylation and total protein by using western blot. We further introduce a GFP-labeling method to monitor SCAP trafficking using confocal microscopy. This protocol can be used in regular biology laboratories.