我们感谢Zehavit Frenkel和桑德拉Tolchinsky技术援助。有关这项工作的研究是由以色列科学基金会（1229年至1207年），德国和以色列的项目合作（DIP - DFG）的赠款支持。

以下协议的目的是为分析总糖蛋白或利益的特定糖蛋白的糖链。这个过程基本上是与整个协议内的几个改变这两种情况下相同。 <ol><li>对于N -连接聚糖代谢的标签需要使用哺乳动物细胞生长在一个90毫米，每个样品菜过夜subconfluent的文化（样本可能代表不同的时间点或治疗）。此协议是NIH 3T3细胞进行了优化。 </li><li>饿死孵育5毫升新鲜配制的预热（37℃）葡萄糖的培养基，提供10％的透析FCS和丙酮酸钠4毫米为5-15分钟，对葡萄糖的细胞。 </li><li>饥饿介质更换1毫升预热（37 ° C）葡萄糖免费培养基中含有400μCi[2 - 3 H] -标记的甘露糖（低浓度65μCi/ ml的总糖蛋白标签），孵育的细胞在正常组织培养条件下，1小时。 </li><li>从每个样品中取出的标签介质，并仔细添加2毫升的PBS在4 ° C到相应的脉冲，并放置在冰（这些样本简称为脉冲）的样品，而追逐相应的样品需要漂洗3次，用2 ml预热（37 ° C）正常完整的培养液中，然后放置在CO 2培养箱在37 ° C预热所需的追逐期间的常规培养基用5毫升。 </li><li> 2毫升冰冷的PBS冲洗脉冲样本（以前置于冰上）的3倍。细胞，然后刮掉在2毫升PBS盘使用细胞刮刀，并放置到2 mL Eppendorf管。 </li><li>短自旋16000Xg（6-10秒）的细胞，弃去上清液，并冻结细胞沉淀于-80 ° C。 </li><li>执行步骤5和6在追逐时间结束的追逐样品。 </li><li>从冰箱中取出的细胞和300缓冲液溶解，涡旋简要孵化冰20分钟，或在分析总糖蛋白的情况下孵育缓冲液B冰20分钟，其次是超声（4次，10秒，最大振幅），再煮沸5分钟的样品。 </li><li>在16000Xg离心的细胞裂解液，在4 ° C 20分钟将上清转移至一个新的Eppendorf管中，弃去沉淀。 </li><li>如果分析总糖蛋白，进行分子过滤和deglycosylation（17步）。对于这里的H2A糖蛋白的免疫证明，添加20l/sample（Repligen）蛋白A -琼脂糖珠1:1暂停和3μL/兔抗H2A所需的糖蛋白抗体的样本（在我们的例子反H2A抗体），从第9步上清。 </li><li>样品在4 ° C孵育4-16小时，不断缓慢旋转混合。 </li><li> 30秒，在16000Xg离心珠，小心取出上清液用真空。 </li><li>珠洗净，加入500μL缓冲液D和震荡。旋在第12步，去除上清。重复洗3次。 </li><li>新增加入10μl变性缓冲液（NEB远藤- H的套件提供）珠沉淀，煮沸5分钟（总蛋白分析时，deglycosylation上进行过滤器的单片机，​​第17步）的样品。 </li><li>离心珠（16000Xg 30秒），并转移到一个新的Eppendorf管中的上清液。远藤H酶0.5μL加入到每个样品10μL的反应缓冲液（与NEB远藤H套件还提供），样品在37 ° C为3小时。 </li><li>要单独释放的蛋白质聚糖，样品用去离子水稀释5倍，并放置一个分子过滤器（单片机Ultracel YM30）上，用一个30kDa截止3分钟，然后在14000Xg离心。应用去离子水，并重复离心样品50μL。这冲洗，重复2次以上，保持流过。 </li><li>如果要分析总糖蛋白，适用于单片机过滤的细胞裂解液上清（从第9步）。提取液与100μL缓冲区E，3次重复14000Xg离心3分钟清洗。加入3μL10X远藤H的反应缓冲液和1.5μL远藤H酶单片机截留，孵育3小时37 ° C。 3反复离心发布聚糖与去离子水洗脱，在第16步。 </li><li>如果需要的话，retentates步骤16和17，含有抗远藤的H -糖蛋白的N -糖苷酶F 200亩，然后孵育15μL缓冲液C，37 ° 16小时彗星用去离子水洗脱是在第16步完成。 </li><li>将管中，在Speed​​Vac浓缩流过的远藤H的反应（步骤16和17），干燥完全的样品（这是可以做到高达45 ° C至加速这一进程）。 </li><li>要准备的高效液相色谱法样品，重悬在12μL的HPLC溶剂（乙腈：水，60:40，1％磷酸）的干燥颗粒。 </li><li> HPLC设备（连接到Spherisorb列）来调整流量恒定的溶剂（1毫升/分钟）和压力（1000和2000 psi之间的一个特定值），并放置一小部分收集器，以改变管每隔1分钟。 </li><li>装入样品的HPLC设备，并同时启动馏分收集。 </li><li>收集从每个样品运行，并从一个标准的低聚糖混合物运行的48 1毫升的分数。 </li><li>从每个几分闪烁瓶，转移500μL和3毫升水混合闪烁液混合使用的内容。 </li><li>装入小瓶和闪烁计数器中读取。 </li><li> CPM的读数绘制成的一小部分数量的函数。 </li><li>在高峰期的CPM值代表一个具体聚糖物种如下：分数18-21对应M5的22日至26日到M6，分数，分数27-31 M7的，分数32-35 M8的36-39以M9的，分数，分数40-42 G1M9和分数43-45 G2M9。 </li><li> cpm值在每个峰的分数的总和，体现了一个具体聚糖品种的绝对量。为了弥补物种中含有较多的甘露糖残基的事实，获取更多的标签，每个聚糖物种“相对摩尔量”计算公式如下：每个聚糖物种的绝对量是由甘露糖残基的数目，它包含。然后将这个值除以获得每个具体聚糖品种获得“总数的百分之”的所有聚糖物种的相对摩尔金额的总和。 </li></ol><table border="1"><tbody><tr><td style="text-align:center;">大通（H） </td><td style="text-align:center;"> 0 </td><td style="text-align:center;"> 4 </td></tr><tr><td>发表远藤H（CPM） 一个 </td><td> 90000 </td><td> 16800 </td></tr><tr><td>发布的N -糖苷酶F（CPM）B </td><td> 20400 </td><td> 3900 </td></tr><tr><td>糖蛋白在截留（CPM）C </td><td> 371700 </td><td> 127695 </td></tr><tr><td> Dolichol寡糖截留（CPM）D </td><td> 4350 </td><td> 540 </td></tr><tr><td>截留Dolichol低聚糖（％）Ë </td><td> 1.4 ± 0.7 </td><td> 0.2 ± 0.2 </td></tr></tbody></table> 表1分析deglycosidases和N -连接的糖蛋白样品dolichol寡糖的存在的糖链的释放。 NIH 3T3细胞裂解液从我们应用脉冲追逐过程。单片机过滤后，标记的N -连接寡糖主要发布与远藤H脉冲标记后（高甘露糖型）和进一步治疗的N -糖苷酶F追逐期后，将对应的复杂类型（高尔基体加工多聚糖），远藤H耐低聚糖。这种定量分析确定，dolichol寡糖，占微不足道的一小部分的标签在初始截留。 注： <ol class="lalpha"><li>脉冲[2 - 3 H]标记的甘露糖和NIH 3T3细胞完全培养基追。孵育细胞裂解和过滤通过单片机YM30后，被释放的高甘露糖N -连接寡糖与远藤H.从retentates </li><li>远藤抗H -（一）单片机retentates的材料，然后培养的N -糖苷酶F，并公布在闪烁计数器测量的低聚糖。 </li><li>单片机retentates获得（一）远藤H处理前，用氯仿提取4次：甲醇：水10点10分03秒，以消除易制毒化学糖脂，unextracted材料1N NaOH溶液溶解，并在闪烁计数器计数。 </li><li> Dolichol寡糖纯化提取物（三）所述的闪烁计数器计数。 </li><li>平均3％dolichol寡糖总截留CPM（D）实验（C + D）。 </li></ol>代表性的成果<img src="/files/ftp_upload/1899/1899fig1.jpg" alt="图1" /> 图1。 1h后的脉冲追逐总NIH在未经处理的细胞，并经蛋白酶抑制3T3糖蛋白分析与脉冲标签。[2 - 3 H]，我们取得了一些glucosylated预期的个人资料余下的前体（GLC 2人9 2（G2M9）葡萄糖和G1M9），但大部分标签在M9和M8种，后者是修剪所有的葡萄糖和甘露糖残基（图1）的结果。没有自由的甘露糖或其他小前体进行了检测，证明彻底的净化。经过一个相当长的8H追逐有更广泛的修整M7 - 6和少量的M5，但主要品种继续M​​9和M8（图一B）。如果追一个蛋白酶抑制剂（30微米MG - 132）的存在，只是有轻微的修剪这些相同的物种积累（图1 C）。 <img src="/files/ftp_upload/1899/1899fig2.jpg" alt="图2" /> 图2。相比，总糖蛋白糖链的ERAD基板的定量分析 。 （一）在一项实验中类似图1中，每个低聚糖物种的相对摩尔金额计算，甘露糖含量。 （二）我们每个聚糖物种获得的cpm值转换，每个物种，相对的所有物种目前的相对摩尔金额总和的百分比作为追逐的时间平均两个实验的功能，然后绘制。类似到（a）除ERAD基板ASGPR H2A发布的聚糖分析后，到4H脉冲标记和追逐中存在的蛋白酶抑制剂MG - 132的情况下，（C）值4H追逐的存在MG - 132（A）和（B）是比ASGPR H2A和糖蛋白池（四）修剪在ER进程的N -连接糖链的计划。糖修整过程，导致M6 - 5蛋白（R）的，有针对性地对这些相比，M9 - 8 ERAD，出口到高尔基体和超越。结果表明，ERAD的过程是修剪N -糖链的甘露糖产量与剩余的5-6甘露糖残基物种。

<ol>
	<li>Parodi, A. J., Behrens, N. H., Leloir, L. F., Carminatti, H. <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+polyprenol-bound+saccharides+as+intermediates+in+glycoprotein+synthesis+in+liver.">The role of polyprenol-bound saccharides as intermediates in glycoprotein synthesis in liver.</a> Proc Natl Acad Sci U S A. 69 (11), 3268-3272 (1972).</li><li>Avezov, E., Frenkel, Z., Ehrlich, M., Herscovics, A., Lederkremer, G. Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endoplasmic+reticulum+(ER)+mannosidase+I+is+compartmentalized+and+required+for+N-glycan+trimming+to+Man5-6GlcNAc2+in+glycoprotein+ER-associated+degradation.">Endoplasmic reticulum (ER) mannosidase I is compartmentalized and required for N-glycan trimming to Man5-6GlcNAc2 in glycoprotein ER-associated degradation.</a> Mol Biol Cell. 19 (1), 216-225 (2008).</li><li>Frenkel, Z., Gregory, W., Kornfeld, S., Lederkremer, G. Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endoplasmic+reticulum-associated+degradation+of+mammalian+glycoproteins+involves+sugar+chain+trimming+to+Man6-5GlcNAc2.">Endoplasmic reticulum-associated degradation of mammalian glycoproteins involves sugar chain trimming to Man6-5GlcNAc2.</a> J Biol Chem. 278 (36), 34119-34124 (2003).</li><li>Hosokawa, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhancement+of+endoplasmic+reticulum+(ER)+degradation+of+misfolded+Null+Hong+Kong+alpha1-antitrypsin+by+human+ER+mannosidase+I.">Enhancement of endoplasmic reticulum (ER) degradation of misfolded Null Hong Kong alpha1-antitrypsin by human ER mannosidase I.</a> J Biol Chem. 278 (28), 26287-26294 (2003).</li><li>Jakob, C. A., Burda, P., Roth, J., Aebi, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Degradation+of+misfolded+endoplasmic+reticulum+glycoproteins+in+Saccharomyces+cerevisiae+is+determined+by+a+specific+oligosaccharide+structure.">Degradation of misfolded endoplasmic reticulum glycoproteins in Saccharomyces cerevisiae is determined by a specific oligosaccharide structure.</a> J Cell Biol. 142 (5), 1223-1233 (1998).</li><li>Tolchinsky, S., Yuk, M. H., Ayalon, M., Lodish, H. F., Lederkremer, G. Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Membrane-bound+versus+secreted+forms+of+human+asialoglycoprotein+receptor+subunits.+Role+of+a+juxtamembrane+pentapeptide.">Membrane-bound versus secreted forms of human asialoglycoprotein receptor subunits. Role of a juxtamembrane pentapeptide.</a> J Biol Chem. 271 (24), 14496-14503 (1996).</li></ol>

HPLC分离的活细胞聚糖的脉冲追逐分析提供了一个学习生活的一种糖蛋白，低聚糖的结构上的改动动态方法。有越来越多的证据表明，这样的改变是在参与生产的信号ER折叠，质量控制和贩运系统 2-5 。不仅对一个特定的糖蛋白的利益，但也总糖蛋白，如本文所示，我们比较一个特定的ERAD基板对比的命运和细胞糖蛋白池的结构动力学分析，该方法可应用于。净化技术是简单但却十分具体。?...

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		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>600E Multisolvent delivery System controller</td>
<td>&nbsp;</td>
<td>Waters</td>
<td>WAT062710</td>
<td>&nbsp;</td>
<tr>
<td>Acetonitrile LiChrosolv (gradient grade for liquid chromatography)</td>
<td>&nbsp;</td>
<td>Merck Bioscience</td>
<td>1.0003</td>
<td>&nbsp;</td>
<tr>
<td>Microcon Amicon Ultra 0.5 ml 30K or Centricon ultracel YM-30</td>
<td>&nbsp;</td>
<td>Millipore</td>
<td>UFC503024 or 4208</td>
<td>&nbsp;</td>
<tr>
<td>Concentrator 5301, incl. 48 x 1.5 / 2.0 ml fixed-angle rotor</td>
<td>&nbsp;</td>
<td>Eppendorf</td>
<td>5301 000.016</td>
<td>&nbsp;</td>
<tr>
<td>Dialyzed Foetal Calf Serum</td>
<td>&nbsp;</td>
<td>Biological Industries</td>
<td>04-011-1A</td>
<td>&nbsp;</td>
<tr>
<td>Dulbecco's Modi&#xFB01;ed Eagle's Medium</td>
<td>&nbsp;</td>
<td>Gibco Invitrogen Cell Culture</td>
<td>41965039</td>
<td>&nbsp;</td>
<tr>
<td>Dulbecco&#x2019;s Modi&#xFB01;ed Eagle's Medium - glucose free</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>D5030</td>
<td>&nbsp;</td>
<tr>
<td>Dulbecco's Phosphate Buffered Saline (PBS)</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>D1408</td>
<td>&nbsp;</td>
<tr>
<td>EDTA disodium salt (Titriplex Iii)</td>
<td>&nbsp;</td>
<td>Merck</td>
<td>1084180250</td>
<td>&nbsp;</td>
<tr>
<td>Endo Hf Kit (1,000,000 units/ml)</td>
<td>&nbsp;</td>
<td>New England Biolabs</td>
<td>p0703S</td>
<td>&nbsp;</td>
<tr>
<td>Foetal Calf Serum (FCS)</td>
<td>&nbsp;</td>
<td>Biological Industries</td>
<td>04-001-1A</td>
<td>&nbsp;</td>
<tr>
<td>Frac-100 fraction collector</td>
<td>&nbsp;</td>
<td>Amersham Biosciences</td>
<td>18-1000-77</td>
<td>&nbsp;</td>
<tr>
<td>LS 6500 Liquid Scintillation Counting systems</td>
<td>&nbsp;</td>
<td>Beckman Coulter</td>
<td>510720</td>
<td>&nbsp;</td>
<tr>
<td>Mannose, D-[2-3H(N)]- (Specific Activity: 15-30Ci/mmol)</td>
<td>&nbsp;</td>
<td>PerkinElmer Life Sciences</td>
<td>NET570A</td>
<td>&nbsp;</td>
<tr>
<td>N-carbobenzoxyl-leucinyl-leucinyl-leucinal (MG-132)</td>
<td>&nbsp;</td>
<td>Calbiochem</td>
<td>474790</td>
<td>&nbsp;</td>
<tr>
<td>N-glycosidase F (1000 units/ml)</td>
<td>&nbsp;</td>
<td>Roche</td>
<td>11365177</td>
<td>&nbsp;</td>
<tr>
<td>N-octylglucoside</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>O3757</td>
<td>&nbsp;</td>
<tr>
<td>NIH 3T3 cells</td>
<td>&nbsp;</td>
<td>American Type Culture Collection (ATCC)</td>
<td>CRL-1658</td>
<td>&nbsp;</td>
<tr>
<td>Opti-Flour</td>
<td>&nbsp;</td>
<td>PerkinElmer Life Sciences</td>
<td>6013199</td>
<td>&nbsp;</td>
<tr>
<td>Phosphoric acid solution ( 49-51%, for HPLC)</td>
<td>&nbsp;</td>
<td>Fluka</td>
<td>79607</td>
<td>&nbsp;</td>
<tr>
<td>Protease Inhibitor Cocktail</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>P2714</td>
<td>&nbsp;</td>
<tr>
<td>Protein A-Sepharose beads</td>
<td>&nbsp;</td>
<td>Repligen</td>
<td>IPA300</td>
<td>&nbsp;</td>
<tr>
<td>Rabbit polyclonal anti-H2 carboxy-terminal 6</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Sodium deoxycholate</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>30970</td>
<td>&nbsp;</td>
<tr>
<td>Sodium dodecyl sulfate</td>
<td>&nbsp;</td>
<td>Bio-RAD</td>
<td>161-0301</td>
<td>&nbsp;</td>
<tr>
<td>Sodium phosphate</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>342483</td>
<td>&nbsp;</td>
<tr>
<td>Sodium pyruvate solution (100mM)</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>S8636</td>
<td>&nbsp;</td>
<tr>
<td>Spherisorb NH2 Column, 5 &mu;m, 4.6 x 250 mm</td>
<td>&nbsp;</td>
<td>Waters</td>
<td>PSS831115</td>
<td>&nbsp;</td>
<tr>
<td>Triton X-100</td>
<td>&nbsp;</td>
<td>BDH</td>
<td>306324N</td>
<td>&nbsp;</td>
<tr>
<td>Vibra-Cell ultrasonic processors VCX 750</td>
<td>&nbsp;</td>
<td>Sonics and Materials, Inc.</td>
<td>690-003</td>
<td>&nbsp;</td>
<tr>
<td>Buffer A: 1% Triton X-100, 0.5% w/v sodium deoxycholate, Protease inhibitor cocktail 2% v/v in PBS</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Buffer B: 0.5% w/v SDS, Protease inhibitor cocktail 2% v/v in PBS</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Buffer C: N-glycosidase F reaction buffer: 200mM Na3PO4, pH 8.0, 4mM EDTA, 1.5% N-octylglucoside</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>NIH 3T3 stably expressing H2a 6</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Buffer D: 0.5% Triton X-100, 0.25% w/v sodium deoxycholate, 0.5% w/v SDS, Protease inhibitor cocktail 2% v/v in PBS</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Buffer E: 0.5% w/v SDS, 1% v/v 2-Mercaptoethanol, Protease inhibitor cocktail 2% v/v in PBS </td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>2-mercaptoethanol</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>M3148</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

pulse-chase analysis of n-linked sugar chains from glycoproteins in mammalian cells

扣押的GLC<sub&gt; 3</sub&gt;男<sub&gt; 9</sub&gt;葡萄糖<sub&gt; 2</sub&gt;易制毒化学寡糖在ER新生多肽是一种分泌蛋白的共同修改。虽然这种修改是在多种生物过程中有牵连的其他方面，它的功能不断涌现，其生产糖蛋白的质量控制和贩运的信号的作用最近的证据。因此，相关的N -连接聚糖及其加工的现象正在紧锣密鼓地调查。最近已开发的蛋白质组学分析的方法，大大提高了N -连接糖蛋白聚糖的特性。然而，他们并没有提供洞察入的糖链参与处理的动态。对于这一点，标签和使用脉冲追逐分析协议，通常是复杂的，并给予非常低的收益率。我们在这里介绍一个简单的方法代谢为N -连接寡糖的分离和分析。该协议是基于标签的细胞[2 -<sup&gt; 3</sup&gt; H]甘露糖，变性裂解和低聚糖的酶释放，无论是从利益特别是免疫沉淀蛋白质，或从远藤H和N -糖苷酶F分子过滤（Amicon），序贯疗法与一般的糖蛋白池。在这个方法中分离低聚糖的高效液相色谱分析的输入，它允许不同的糖链结构之间的歧视，包括他们的单糖单位的数量，作为一个单糖的决议。使用这种方法，我们能够研究后，在正常情况下的脉冲追逐下蛋白酶抑制高甘露糖总细胞糖蛋白的N -连接寡糖型材。这些配置文件进行了比较，从免疫沉淀ER相关的降解（ERAD）衬底上获得的。我们的研究结果表明，大多数的NIH 3T3细胞糖蛋白是相对稳定的，他们低聚糖大多是修剪到人<sub&gt; 9-8</sub&gt;葡萄糖<sub&gt; 2</sub&gt;。相反，不稳定的ERAD基板修剪到人<sub&gt; 6-5</sub&gt;葡萄糖<sub&gt; 2</sub&gt;和糖蛋白轴承这些物种的积累后，抑制蛋白酶体降解。

Watch this Scientific Journal Video about Pulse-chase Analysis of N-linked Sugar Chains from Glycoproteins in Mammalian Cells at JoVE.com

Pulse-chase Analysis of N-linked Sugar Chains from Glycoproteins in Mammalian Cells

我们描述了一种通过改变早期的生活后，其在哺乳动物细胞中合成的糖蛋白的N -连接聚糖分析方法。这是通过分析代谢标记聚糖，从糖蛋白和高效液相色谱法检查酶的释放脉冲追逐。

N -连接糖链的糖蛋白在哺乳动物细胞中的脉冲大通分析

Attachment of the Glc3Man9GlcNAc2 precursor oligosaccharide to nascent polypeptides in the ER is a common modification for secretory proteins. Although ...

pulse-chase-analysis-n-linked-sugar-chains-from-glycoproteins

Research

JoVE Journal

Biology

15.1K 次观看.  Tel Aviv University. 我们描述了一种通过改变早期的生活后，其在哺乳动物细胞中合成的糖蛋白的N -连接聚糖分析方法。这是通过分析代谢标记聚糖，从糖蛋白和高效液相色谱法检查酶的释放脉冲追逐。

视频: Pulse-chase Analysis of N-linked Sugar Chains from Glycoproteins in Mammalian Cells

In situ Subcellular Fractionation of Adherent and Non-adherent Mammalian Cells

Protein function is intimately coupled to protein localization. Although some proteins are restricted to a specific location or subcellular compartment, many proteins are present as a freely diffusing population in free exchange with a sub-population that is tightly associated with a particular subcellular domain or structure. In situ subcellular fractionation allows the visualization of protein compartmentalization and can also reveal protein sub-populations that localize to specific structures. For example, removal of soluble cytoplasmic proteins and loosely held nuclear proteins can reveal the stable association of some transcription factors with chromatin. Subsequent digestion of DNA can in some cases reveal association with the network of proteins and RNAs that is collectively termed the nuclear scaffold or nuclear matrix. 
Here we describe the steps required during the in situ fractionation of adherent and non-adherent mammalian cells on microscope coverslips. Protein visualization can be achieved using specific antibodies or fluorescent fusion proteins and fluorescence microscopy. Antibodies and/or fluorescent dyes that act as markers for specific compartments or structures allow protein localization to be mapped in detail. In situ fractionation can also be combined with western blotting to compare the amounts of protein present in each fraction. This simple biochemical approach can reveal associations that would otherwise remain undetected.

In situ Subcellular Fractionation of Adherent and Non-adherent Mammalian Cells

In situ subcellular fractionation of mammalian cells on microscope coverslips allows the visualisation of protein localisation.

原位贴壁和非贴壁的哺乳动物细胞的亚细胞分馏

Protein function is intimately coupled to protein localization. Although some proteins are restricted to a specific location or subcellular compartment, ...

科研

生物学

Analysis of DNA Double-strand Break (DSB) Repair in Mammalian Cells

DNA double-strand breaks are the most dangerous DNA lesions that may lead to massive loss of genetic information and cell death. Cells repair DSBs using two major pathways: nonhomologous end joining (NHEJ) and homologous recombination (HR). Perturbations of NHEJ and HR are often associated with premature aging and tumorigenesis, hence it is important to have a quantitative way of measuring each DSB repair pathway. Our laboratory has developed fluorescent reporter constructs that allow sensitive and quantitative measurement of NHEJ and HR. The constructs are based on an engineered GFP gene containing recognition sites for a rare-cutting I-SceI endonuclease for induction of DSBs. The starting constructs are GFP negative as the GFP gene is inactivated by an additional exon, or by mutations. Successful repair of the I-SceI-induced breaks by NHEJ or HR restores the functional GFP gene. The number of GFP positive cells counted by flow cytometry provides quantitative measure of NHEJ or HR efficiency.

Analysis of DNA Double-strand Break DSB Repair in Mammalian Cells

This article describes GFP-based fluorescence in vivo assays that separately quantify homologous recombination and nonhomologous end joining in mammalian cells.

在哺乳动物细胞中的DNA双链断裂（DSB）的维修分析

DNA double-strand breaks are the most dangerous DNA lesions that may lead to massive loss of genetic information and cell death.  Cells repair DSBs using ...

MISSION esiRNA for RNAi Screening in Mammalian Cells

RNA interference (RNAi) is a basic cellular mechanism for the control of gene expression. RNAi is induced by short double-stranded RNAs also known as small interfering RNAs (siRNAs). The short double-stranded RNAs originate from longer double stranded precursors by the activity of Dicer, a protein of the RNase III family of endonucleases. The resulting fragments are components of the RNA-induced silencing complex (RISC), directing it to the cognate target mRNA. RISC cleaves the target mRNA thereby reducing the expression of the encoded protein1,2,3. RNAi has become a powerful and widely used experimental method for loss of gene function studies in mammalian cells utilizing small interfering RNAs.
Currently two main methods are available for the production of small interfering RNAs. One method involves chemical synthesis, whereas an alternative method employs endonucleolytic cleavage of target specific long double-stranded RNAs by RNase III in vitro. Thereby, a diverse pool of siRNA-like oligonucleotides is produced which is also known as endoribonuclease-prepared siRNA or esiRNA. A comparison of efficacy of chemically derived siRNAs and esiRNAs shows that both triggers are potent in target-gene silencing. Differences can, however, be seen when comparing specificity. Many single chemically synthesized siRNAs produce prominent off-target effects, whereas the complex mixture inherent in esiRNAs leads to a more specific knockdown10.
In this study, we present the design of genome-scale MISSION esiRNA libraries and its utilization for RNAi screening exemplified by a DNA-content screen for the identification of genes involved in cell cycle progression. We show how to optimize the transfection protocol and the assay for screening in high throughput. We also demonstrate how large data-sets can be evaluated statistically and present methods to validate primary hits. Finally, we give potential starting points for further functional characterizations of validated hits.

Here we use a human esiRNA library in a high-throughput screen for genes involved in cell division. We demonstrate how to set up and conduct an esiRNA screens, as well as how to analyze and validate the results.

在哺乳动物细胞的RNAi筛选使命esiRNA

RNA interference (RNAi) is a basic cellular mechanism for the control of gene expression. RNAi is induced by short double-stranded RNAs also known as ...

Mutagenesis and Functional Analysis of Ion Channels Heterologously Expressed in Mammalian Cells

We will demonstrate how to study the functional effects of introducing a point mutation in an ion channel. We study G protein-gated inwardly rectifying potassium (referred to as GIRK) channels, which are important for regulating the excitability of neurons. There are four different mammalian GIRK channel subunits (GIRK1-GIRK4) - we focus on GIRK2 because it forms a homotetramer. Stimulation of different types of G protein-coupled receptors (GPCRs), such as the muscarinic receptor (M2R), leads to activation of GIRK channels. Alcohol also directly activates GIRK channels. We will show how to mutate one amino acid by specifically changing one or more nucleotides in the cDNA for the GIRK channel. This mutated cDNA sequence will be amplified in bacteria, purified, and the presence of the point mutation will be confirmed by DNA sequencing. The cDNAs for the mutated and wild-type GIRK channels will be transfected into human embryonic kidney HEK293T cells cultured in vitro. Lastly, whole-cell patch-clamp electrophysiology will be used to study the macroscopic potassium currents through the ectopically expressed wild-type or mutated GIRK channels. In this experiment, we will examine the effect of a L257W mutation in GIRK2 channels on M2R-dependent and alcohol-dependent activation.

We will demonstrate how to study the effect of a single point mutation on the function of an ion channel.

异源表达在哺乳动物细胞离子通道的突变和功能分析

We will demonstrate how to study the functional effects of introducing a point mutation in an ion channel. We study G protein-gated inwardly rectifying ...

Analysis of mRNA Nuclear Export Kinetics in Mammalian Cells by Microinjection

In eukaryotes, messenger RNA (mRNA) is transcribed in the nucleus and must be exported into the cytoplasm to access the translation machinery. Although the nuclear export of mRNA has been studied extensively in Xenopus oocytes1 and genetically tractable organisms such as yeast2 and the Drosophila derived S2 cell line3, few studies had been conducted in mammalian cells. Furthermore the kinetics of mRNA export in mammalian somatic cells could only be inferred indirectly4,5. In order to measure the nuclear export kinetics of mRNA in mammalian tissue culture cells, we have developed an assay that employs the power of microinjection coupled with fluorescent in situ hybridization (FISH). These assays have been used to demonstrate that in mammalian cells, the majority of mRNAs are exported in a splicing dependent manner6,7, or in manner that requires specific RNA sequences such as the signal sequence coding region (SSCR) 6. In this assay, cells are microinjected with either in vitro synthesized mRNA or plasmid DNA containing the gene of interest. The microinjected cells are incubated for various time points then fixed and the sub-cellular localization of RNA is assessed using FISH. In contrast to transfection, where transcription occurs several hours after the addition of nucleic acids, microinjection of DNA or mRNA allows for rapid expression and allows for the generation of precise kinetic data.

Here we describe an assay that employs the power of microinjection coupled with fluorescent in situ hybridization in order to accurately measure the nuclear export kinetics of mRNA in mammalian somatic cells.

在哺乳动物细胞通过显微注射mRNA的核出口动力学分析

In eukaryotes, messenger RNA (mRNA) is transcribed in the nucleus and must be exported into the cytoplasm to access the translation machinery. Although ...

Analysis of Cell Cycle Position in Mammalian Cells

The regulation of cell proliferation is central to tissue morphogenesis during the development of multicellular organisms. Furthermore, loss of control of cell proliferation underlies the pathology of diseases like cancer. As such there is great need to be able to investigate cell proliferation and quantitate the proportion of cells in each phase of the cell cycle. It is also of vital importance to indistinguishably identify cells that are replicating their DNA within a larger population. Since a cell&prime;s decision to proliferate is made in the G1 phase immediately before initiating DNA synthesis and progressing through the rest of the cell cycle, detection of DNA synthesis at this stage allows for an unambiguous determination of the status of growth regulation in cell culture experiments.
DNA content in cells can be readily quantitated by flow cytometry of cells stained with propidium iodide, a fluorescent DNA intercalating dye. Similarly, active DNA synthesis can be quantitated by culturing cells in the presence of radioactive thymidine, harvesting the cells, and measuring the incorporation of radioactivity into an acid insoluble fraction. We have considerable expertise with cell cycle analysis and recommend a different approach. We Investigate cell proliferation using bromodeoxyuridine/fluorodeoxyuridine (abbreviated simply as BrdU) staining that detects the incorporation of these thymine analogs into recently synthesized DNA. Labeling and staining cells with BrdU, combined with total DNA staining by propidium iodide and analysis by flow cytometry1 offers the most accurate measure of cells in the various stages of the cell cycle. It is our preferred method because it combines the detection of active DNA synthesis, through antibody based staining of BrdU, with total DNA content from propidium iodide. This allows for the clear separation of cells in G1 from early S phase, or late S phase from G2/M. Furthermore, this approach can be utilized to investigate the effects of many different cell stimuli and pharmacologic agents on the regulation of progression through these different cell cycle phases.
In this report we describe methods for labeling and staining cultured cells, as well as their analysis by flow cytometry. We also include experimental examples of how this method can be used to measure the effects of growth inhibiting signals from cytokines such as TGF-&beta;1, and proliferative inhibitors such as the cyclin dependent kinase inhibitor, p27KIP1. We also include an alternate protocol that allows for the analysis of cell cycle position in a sub-population of cells within a larger culture5. In this case, we demonstrate how to detect a cell cycle arrest in cells transfected with the retinoblastoma gene even when greatly outnumbered by untransfected cells in the same culture. These examples illustrate the many ways that DNA staining and flow cytometry can be utilized and adapted to investigate fundamental questions of mammalian cell cycle control.

Determining the cell cycle position of a population of cells, or understanding how signals affect proliferation, can be readily measured by flow cytometry using this protocol. We report a simple experimental approach to staining cells and quantifying their position in the cell cycle.

在哺乳动物细胞中细胞周期位置分析

The regulation of cell proliferation is central to tissue morphogenesis during the development of multicellular organisms. Furthermore, loss of control of ...

Expression Analysis of Mammalian Linker-histone Subtypes

Linker histone H1 binds to the nucleosome core particle and linker DNA, facilitating folding of chromatin into higher order structure. H1 is essential for mammalian development1 and regulates specific gene expression in vivo2-4. Among the highly conserved histone proteins, the family of H1 linker histones is the most heterogeneous group. There are 11 H1 subtypes in mammals that are differentially regulated during development and in different cell types. These H1 subtypes include 5 somatic H1s (H1a-e), the replacement H10, 4 germ cell specific H1 subtypes, and H1x5. The presence of multiple H1 subtypes that differ in DNA binding affinity and chromatin compaction ability6-9 provides an additional level of modulation of chromatin function. Thus, quantitative expression analysis of individual H1 subtypes, both of mRNA and proteins, is necessary for better understanding of the regulation of higher order chromatin structure and function.
Here we describe a set of assays designed for analyzing the expression levels of individual H1 subtypes (Figure 1). mRNA expression of various H1 variant genes is measured by a set of highly sensitive and quantitative reverse transcription-PCR (qRT-PCR) assays, which are faster, more accurate and require much less samples compared with the alternative approach of Northern blot analysis. Unlike most other cellular mRNA messages, mRNAs for most histone genes, including the majority of H1 genes, lack a long polyA tail, but contain a stem-loop structure at the 3' untranslated region (UTR)10. Therefore, cDNAs are prepared from total RNA by reverse transcription using random primers instead of oligo-dT primers. Realtime PCR assays with primers specific to each H1 subtypes (Table 1) are performed to obtain highly quantitative measurement of mRNA levels of individual H1 subtypes. Expression of housekeeping genes are analyzed as controls for normalization. 
The relative abundance of proteins of each H1 subtype and core histones is obtained through reverse phase high-performance liquid chromatography (RP-HPLC) analysis of total histones extracted from mammalian cells11-13. The HPLC method and elution conditions described here give optimum separations of mouse H1 subtypes. By quantifying the HPLC profile, we calculate the relative proportion of individual H1 subtypes within H1 family, as well as determine the H1 to nucleosome ratio in the cells.

We describe a set of assays to analyze expression levels of H1 linker histones. mRNA of individual H1 genes are quantitatively measured by random primer based reverse transcription followed by real-time PCR, whereas protein quantification of H1 histones is achieved by HPLC analysis.

哺乳动物链接组蛋白亚型表达分析

Linker histone H1 binds to the nucleosome core particle and linker DNA, facilitating folding of chromatin into higher order structure.  H1 is essential ...

Visualization of Endoplasmic Reticulum Localized mRNAs in Mammalian Cells

In eukaryotes, most of the messenger RNAs (mRNAs) that encode secreted and membrane proteins are localized to the surface of the endoplasmic reticulum (ER). However, the visualization of these mRNAs can be challenging. This is especially true when only a fraction of the mRNA is ER-associated and their distribution to this organelle is obstructed by non-targeted (i.e. "free") transcripts. In order to monitor ER-associated mRNAs, we have developed a method in which cells are treated with a short exposure to a digitonin extraction solution that selectively permeabilizes the plasma membrane, and thus removes the cytoplasmic contents, while simultaneously maintaining the integrity of the ER. When this method is coupled with fluorescent in situ hybridization (FISH), one can clearly visualize ER-bound mRNAs by fluorescent microscopy. Using this protocol the degree of ER-association for either bulk poly(A) transcripts or specific mRNAs can be assessed and even quantified. In the process, one can use this assay to investigate the nature of mRNA-ER interactions.

Here we describe a method to visualize endoplasmic reticulum-associated mRNAs in mammalian tissue culture cells. This technique involves the selective permeabilization of the plasma membrane with digitonin to remove cytoplasmic contents followed by fluorescent in situ hybridization to detect either bulk poly(A) mRNA or specific transcripts.

在哺乳动物细胞中的内质网定位的mRNA的可视化

In eukaryotes, most of the messenger RNAs (mRNAs) that encode secreted and membrane proteins are localized to the surface of the endoplasmic reticulum ...

Induction and Analysis of Epithelial to Mesenchymal Transition

Epithelial to mesenchymal transition (EMT) is essential for proper morphogenesis during development. Misregulation of this process has been implicated as a key event in fibrosis and the progression of carcinomas to a metastatic state. Understanding the processes that underlie EMT is imperative for the early diagnosis and clinical control of these disease states. Reliable induction of EMT in vitro is a useful tool for drug discovery as well as to identify common gene expression signatures for diagnostic purposes. Here we demonstrate a straightforward method for the induction of EMT in a variety of cell types. Methods for the analysis of cells pre- and post-EMT induction by immunocytochemistry are also included. Additionally, we demonstrate the effectiveness of this method through antibody-based array analysis and migration/invasion assays.

A straightforward method is described for the induction of epithelial to mesenchymal transition (EMT) in a variety of cell types. Methods for the analysis of cells in EMT states by immunocytochemistry are included.

诱导上皮和分析间质转化

Epithelial  to mesenchymal transition (EMT) is essential for proper morphogenesis during  development. Misregulation of this  process has been implicated ...

Measurement of Heme Synthesis Levels in Mammalian Cells

Heme serves as the prosthetic group for a wide variety of proteins known as hemoproteins, such as hemoglobin, myoglobin and cytochromes. It is involved in various molecular and cellular processes such as gene transcription, translation, cell differentiation and cell proliferation. The biosynthesis levels of heme vary across different tissues and cell types and is altered in diseased conditions such as anemia, neuropathy and cancer. This technique uses [4-14C] 5-aminolevulinic acid ([14C] 5-ALA), one of the early precursors in the heme biosynthesis pathway to measure the levels of heme synthesis in mammalian cells. This assay involves incubation of cells with [14C] 5-ALA followed by extraction of heme and measurement of the radioactivity incorporated into heme. This procedure is accurate and quick. This method measures the relative levels of heme biosynthesis rather than the total heme content. To demonstrate the use of this technique the levels of heme biosynthesis were measured in several mammalian cell lines.

Altered intracellular heme levels are associated with common diseases such as cancer. Thus, there is a need to measure heme biosynthesis levels in diverse cells. The goal of this protocol is to provide a fast and sensitive method to measure and compare the levels of heme synthesis in different cells.

中血红素的合成水平在哺乳动物细胞中测量

Heme serves as the prosthetic group for a wide variety of proteins known as hemoproteins, such as hemoglobin, myoglobin and cytochromes. It is involved in ...