这项工作是支持的美国国立卫生研究院的VF（NS42599和GM077569）的赠款。

1。准备交联<ol><li>您将需要板足够数量的细胞，让500微克每标准管反应蛋白的隔离。单管可能足以确定一个由免疫印迹的兴趣的蛋白质公认interactors。在这种情况下，珠约束的材料可以用SDS - PAGE样品缓冲液（免疫）洗脱。标准反应质谱分析，应增加至少十倍，应使用的抗体（免疫亲和层析）认可的一种肽类抗原竞争洗脱的蛋白质复合物。这一战略将让免疫球蛋白的蛋白质复合物的隔离。 </li><li>交联的最佳细胞密度是75％至90％汇合。在高汇合条件下，细胞往往桩上对方，从而降低所有细胞的细胞渗透交联剂的无障碍。 </li><li>细胞类型和实验条件下，应标示单元板的底部。我们通常包括仅与车辆控制。 </li><li>除了DSP解决方案，以交联前的所有准备解决方案。 <ol><li>氯化钙2 0.1毫米和氯化钙2 1毫米（PBS / CA /镁）开始实验前准备的磷酸盐缓冲液缓冲库存。除了钙，镁离子是细胞在实验过程中培养板的附着力的关键。保存在4 ° C。 </li><li> 50X完整的蛋白酶抑制剂 - 1ML Milli - Q水溶解1片。储存在-20 ° C。 </li><li> 20％的Triton X - 100的重量了10G TRITON X -100和稀释在50毫升Milli - Q水的总量。岩石在4 ° C过夜，并储存于4 ° C直到使用。不要存放超过1个月。使用这20％的股份稀释，以准备下面描述的缓冲区的裂解和IP。 </li><li> 50X DSP淬火液1M的TRIS pH至7.4。在室温下储存。 </li><li>准备一个10X缓冲的解决方案。 10X缓冲液A： <ul><li> 50毫升1 M羟乙基</li><li> 150毫升5 M氯化钠</li><li> 10毫升0.5 M的EGTA </li><li> 250μL2 M MgCl 2的 </li><li>调整pH值至7.4 </li><li>使终体积为500毫升</li></ul> 10X缓冲液A液稀释至1X缓冲液A的需要。 1X缓冲液A也可用于裂解液和免疫磁性沉淀缓冲区。 缓冲区一个1X <ul><li> 10毫米HEPES </li><li> 150 mM氯化钠</li><li> 1 mM的EGTA </li><li> 0.1毫米氯化镁2 </li><li> pH值7.4 </li></ul></li><li>裂解缓冲液1X缓冲液A + 0.5％Triton X - 100的的。 </li><li>免疫磁性沉淀缓冲液（IP缓冲区）1X缓冲液A + 0.1％Triton X - 100的的。 </li></ol></li></ol> 2。准备交联解决方案<ol><li>立即准备申请前到细胞的DSP解决方案。 DSP是疏水性极强，并应在DMSO溶解在PBS / CA /镁缓冲液稀释。在1 DMSO溶液溶解40毫克的DSP。这使得DSP（DSP分子量是404.42克/摩尔）100毫米的解决方案。 </li><li>适当体积的PBS /钙/镁预热至37 ° C，以便PBS稀释的DSP / DMSO。 各类钢板尺寸所需的卷： 6孔板，每孔2 mL的 10厘米的盘每盘10毫升 15厘米盘每盘20毫升</li><li>新增每1毫升的温暖PBS /钙/镁的DSP / DMSO原液10μL。添加DSP /二甲基亚砜原液下降下降，反复搅拌，直到所有的DSP已经解散。请控制解决方案，每1毫升的PBS /钙/镁添加到10μL二甲基亚砜。 </li><li>放置在一个冰水浴中，超过10分钟不再所有PBS / CA /镁的解决方案。 </li></ol> 3。为交联细胞做准备<ol><li>准备一个冰水浴中，将适合所有板块交联或车辆控制孵化。 </li><li>从37℃培养箱中培养板，并立即放入冰水浴。 </li><li>洗涤细胞两次，用冰冷的PBS / CA /镁。使用相同的音量，你将使用（见2.2节）为交联剂孵化。 </li><li>删除第二次清洗和新增车辆的控制或DSP交联缓冲液中。 </li><li>在冰上培育了两个小时。您应该检查板块大约每隔20分钟，以确保所有细胞都被溶液覆盖。您可能会注意到一个DSP少量沉淀出的解决方案。这是正常的。 </li></ol> 4。灭活DSP的反应<ol><li>准备一个20毫米的Tris pH值7.4的PBS / CA /毫克（每1毫升的PBS /钙/镁20μL的1 M Tris pH值7.4）的1X DSP淬火液。 </li><li>删除对车辆的控制和交联的解决方案。 </li><li>添加冰冷的灭活解决方案，并在冰上孵育15分钟。 </li></ol> 5。细胞裂解<ol><li> DSP淬火孵化期间，准备了0.5％的Triton X - 100的缓冲区中一个完整的蛋白酶抑制剂的鸡尾酒的细胞裂解液。添加完成每1 mL裂解缓冲液20μL50X原液。 </li><li>经过15分钟的DSP淬火孵化，洗涤细胞，用PBS /钙/镁两次。 </li><li> 30分钟，加入裂解液+完整细胞和岩石在4 ° C。有人建议裂解液量为各类板材大小： <ul><li> 6孔板，每孔0.5毫升</li><li> 10厘米的板1毫升每盘</li><li> 15厘米板每板1毫升</li></ul></li><li>裂解后，使用细胞刮刀收集板的细胞。一定要保持在冰上裂解液，以尽量减少蛋白酶活性</li><li>以最快的速度（15.000 XG）在台式迷你离心机旋转细胞裂解液15分钟，在4 ° C。 </li><li>移取上清液到一个新管。弃去沉淀。 </li><li>分析蛋白质水平，并进行免疫。 </li></ol> 6。准备免疫磁性沉淀珠 注意：这一步通常是直接开始后2个小时的交联孵化开始。 <ol><li>结合30μLDYNAL免疫磁珠，IP缓冲区的500μL（1X缓冲液+ 0.1％TRITON X - 100）和适量的抗原特异性抗体screwtop离心管。准备抗体和非特异性抗体管为阴性对照。适量的抗体应确定在不同的实验中，基于个人抗原。标签的所有IP管。 </li><li>允许的IP管，在最终结束两个小时摇杆，在室温孵育。此外，孵化与抗体的IP管一夜之间在4 °一天前交联。 </li><li>孵化后，做一个快速10秒的微型离心收集在试管底部珠。 </li><li>磁持有人，这将拉动磁珠离试管底部滑入管。珠安全的方式，你现在可以轻松洗去任何未结合的抗体。 </li><li>使用凝胶加载提示，或P200提示要删除孵化缓冲区和任何未结合抗体，如小口径抽吸提示。孵化量只要删除添加1毫升IP缓冲区。 </li><li>第管，轻轻地重新暂停的珠子，并在室温下孵育5月底结束旋转分钟的所有IP管。 </li><li>重复的5分钟的清洗步骤（步骤6.3通过6.6）再次与IP缓冲区的新鲜毫升。 </li><li>知识产权管可以留在最后一次洗涤孵化，直到交联裂解液已准备好要加入的珠子。 </li></ol> 7。与免疫磁珠孵育交联裂解液<ol><li>最后洗自旋免疫磁珠后，倒在了10秒的迷你离心机。然后继续滑入磁座管。 </li><li>同样，使用小口径的愿望提示，删除从管的孵化量。立即加入500μL交联的细胞裂解液（假设在1μg/ mL的裂解）含有免疫磁珠管。如果您将使用适量的肽作为阴性对照肽竞争，应包括在这一点上。应执行先前的实验，以确定适当的肽竞争条件。此外，您可能需要有裂解免费免疫磁性珠作为阴性对照。在这种情况下，立即加入500μL的细胞裂解缓冲液（1X缓冲液A + 0.5％TRITON X - 100）+完整的免疫磁珠。 </li><li>轻轻重悬珠。悬浮珠应在4℃孵育在年底结束旋转2小时。 </li></ol> 8。从珠清洗不作承诺裂解液<ol><li>一旦裂解液有足够的孵化时间，做一个快速10日在一个小型离心机的第二个旋转。然后将管进入磁场的持有人。磁持有人应保持在冰浴其余实验。 </li><li>使用小口径抽吸提示，删除所有未绑定的裂解。所有未绑定的裂解液后立即被删除，添加1毫升IP缓冲区。 </li><li>第管，轻轻重悬珠。 </li><li>一旦重悬珠重复珠造粒步骤（8.1）。 </li><li>再次吸了所有的孵化量，并立即加入1毫升IP缓冲区，然后盖管。 </li><li>轻轻重悬在IP缓冲区的珠子。孵育5分钟，在4月底结束摇摆°重悬珠。 </li><li>重复洗涤步骤珠（8.4至8.6）4次以上。 </li></ol> 9。变性样品，收集从珠<ol><li>免疫磁性降水管都被冲走后，再沙珠（8.1）和幻灯片管到磁持有人。 </li><li>绑定的免疫磁珠蛋白在变性条件下被删除。吸了最后一个IP缓冲液洗和删除的磁座的IP管。 </li><li>添加1X凝胶上样缓冲液珠沉淀池珠再次在试管底部的正上方的IP管。样品缓冲液量取决于要使用的凝胶以及大小。您可能需要轻轻敲击试管底部，在凝胶上样缓冲液重悬珠。 </li><li>重复的变性过程（9.3至9.2）为每个IP管。 </li><li>凝胶上样缓冲液悬浮的免疫磁珠，然后加热至75 ° C时5分钟才能完成的变性过程。 </li><li>一旦样品已变性，它可以在一个SDS - PAGE凝胶免疫印迹。清空免疫磁珠可以重新沉淀，离心和从样品磁座中删除。 </li></ol> 10。洗脱的抗原肽交联复合物（免疫亲和层析）。 <ol><li>所有的免疫磁性免疫管后已经洗干净，重新沙珠（8.1）和磁持有人的幻灯片管。吸上清，加10的缓冲液，一个补充用于蛋白质复合物的免疫隔离的抗体识别的抗原肽。您应该测试浓度为有效洗脱从10-200微米不等。 </li><li>育珠2小时，4 ° C。 </li><li>将磁珠在磁场的立场，并仔细收回10毫升洗脱材料。保存这个上清。 </li><li>快速用缓冲液A的珠子和丢弃洗。保存的珠子。 </li><li>凝胶上样缓冲液保存的上清液和珠，加入到1X浓度。这些管子在75℃5分钟孵育。 </li><li>珠和肽洗脱液经SDS - PAGE分析。洗脱液应IgG的蛋白质的SDS - PAGE染色或免疫印迹（图1B）。这种材料的IgG是适合于质谱。 </li></ol><img src="/files/ftp_upload/1855/1855fig1.jpg" alt="图1" /> 图1。 AP - 3相互作用蛋白复合物和膜蛋白的分离 。HEK293细胞（A）或PC12细胞（二）在处理控制车辆的存在（DMSO，奇的车道图1A）或DSP（甚至车道图。 1A或全部车道图1B）。澄清提取物孵育单独珠（图1A，泳道3-4），转铁蛋白受体抗体（图1A，泳道5-6），或AP - 3（图1A，泳道7-10δ抗体;图1B，车道1-10）。免疫复合物，用SDS - PAGE样品缓冲液（图1A）洗脱，一个单独的缓冲区（图1B，泳道1-2），或缓冲δ抗原肽浓度的增加，相应的680 710个氨基酸的补充人类δadaptin（NCBI的：AAD03777; GI：1923266）（图1B，车道3-10）。这种肽结合的δ抗体。肽洗脱后，上清液（S）和珠（二）免疫印迹分析（图1B）。 AP - 3，这是对δ，β3和σ3亚基抗体的检测，可溶性以下因素：网格蛋白重链（CHC），欧盟1亚基pallidin，和啤酒花复杂的亚基vps33b;以及coprecipitates膜蛋白磷酸肌醇- 4 -激酶II型α（PI4KIIα）和锌转运体3（ZnT3）。注意鼠标肽抗体重链的情况下洗脱图上清。 1B。

<ol>
	<li>Salazar, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hermansky-Pudlak+syndrome+protein+complexes+associate+with+phosphatidylinositol+4-kinase+type+II+alpha+in+neuronal+and+non-neuronal+cells.">Hermansky-Pudlak syndrome protein complexes associate with phosphatidylinositol 4-kinase type II alpha in neuronal and non-neuronal cells.</a> J Biol Chem. 284, 1790-1802 (2009).</li><li>Lomant, A. J., Fairbanks, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemical+probes+of+extended+biological+structures:+synthesis+and+properties+of+the+cleavable+protein+cross-linking+reagent+[35S]dithiobis(succinimidyl+propionate.">Chemical probes of extended biological structures: synthesis and properties of the cleavable protein cross-linking reagent [35S]dithiobis(succinimidyl propionate.</a> J Mol Biol. 104, 243-261 (1976).</li><li>Xiang, C. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Using+DSP,+a+reversible+cross-linker,+to+fix+tissue+sections+for+immunostaining,+microdissection+and+expression+profiling.">Using DSP, a reversible cross-linker, to fix tissue sections for immunostaining, microdissection and expression profiling.</a> Nucleic Acids Res. 32, e185-e185 (2004).</li><li>Craige, B., Salazar, G., Faundez, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phosphatidylinositol-4-Kinase+Type+II+Alpha+Contains+an+AP-3+Sorting+Motif+and+a+Kinase+Domain+that+are+both+Required+for+Endosome+Traffic.">Phosphatidylinositol-4-Kinase Type II Alpha Contains an AP-3 Sorting Motif and a Kinase Domain that are both Required for Endosome Traffic.</a> Mol Biol Cell. 19, 1415-1426 (2008).</li><li>Newell-Litwa, K., Seong, E., Burmeister, M., Faundez, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuronal+and+non-neuronal+functions+of+the+AP-3+sorting+machinery.">Neuronal and non-neuronal functions of the AP-3 sorting machinery.</a> J Cell Sci. 120, 531-541 (2007).</li><li>Salazar, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Zinc+Transporter+ZnT3+interacts+with+AP-3+and+it+is+Targeted+to+a+Distinct+Synaptic+Vesicle+Subpopulation.">The Zinc Transporter ZnT3 interacts with AP-3 and it is Targeted to a Distinct Synaptic Vesicle Subpopulation.</a> Mol Biol Cell. 15, 575-587 (2004).</li><li>Trinkle-Mulcahy, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identifying+specific+protein+interaction+partners+using+quantitative+mass+spectrometry+and+bead+proteomes.">Identifying specific protein interaction partners using quantitative mass spectrometry and bead proteomes.</a> J Cell Biol. 183, 223-239 (2008).</li></ol>

DSP，膜渗透，化学还原与12 Å间隔臂的交联剂是用来稳定瞬态蛋白质相互作用 1,2,3,4 。在这里，我们体现这一战略与AP - 3适配器复杂的一种可溶性蛋白复合物，囊泡膜蛋白识别和排序，从内涵体 5 。 AP - 3的选择性结合的锌转运ZnT3和脂质激酶磷酸肌醇4激酶II型阿尔法但不转铁蛋白受体 1,4,6 。我们扩大了这些意见，以确定质谱 1膜蛋白和胞质因素AP - 3蛋白的相互作用...

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		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Phosphate Buffered Saline</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>P4417</td>
<td>Dissolve 1 tablet in 200 mL water; add MgCl2 to a final concentration of 1 mM and CaCl2 to a final concentration of 0.1 mM</td>
</tr>
<tr>
<td>Dithiobis (succinimidyl propionate) (DSP)</td>
<td>&nbsp;</td>
<td>Thermo Scientific</td>
<td>22585</td>
<td>Moisture sensitive, store in air tight container 4&deg;C</td>
</tr>
<tr>
<td>Dimethyl sulphoxide (DMSO) Hybri-Max</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>D2650</td>
<td>&nbsp;</td>
<tr>
<td>Triton X-100, SigmaUltra</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>T9284</td>
<td>&nbsp;</td>
<tr>
<td>Dynabeads, Sheep anti-Mouse IgG</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>110.31</td>
<td>Beads are also available as sheep anti-rabbit</td>
</tr>
<tr>
<td>Dyna-Mag-2 magnet</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>123-21D</td>
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isolation of labile multi-protein complexes by in vivo controlled cellular cross-linking and immuno-magnetic affinity chromatography

蜂窝机械的动态性质经常是建立在瞬态和/或弱蛋白协会。这些低亲和力相互作用排除围绕一个感兴趣的蛋白质，蛋白质网络的隔离和识别的严格方法。使用化学交联剂允许的不稳定相互作用的选择性稳定，从而绕过生化净化的局限性。在这里，我们提出在文化协议细胞适合使用一个homobifunctional交联剂间隔臂12埃，dithiobis（琥珀proprionate）（DSP）。 DSP是一个分子中的二硫键还原裂解。交联与磁珠的利益的蛋白质与免疫层析相结合，使蛋白质复合物，否则将不能承受净化隔离。此协议是与常规免疫印迹技术兼容，它可以扩展为蛋白质鉴定的质谱1。 斯蒂芬妮A. Zlatic和珠江五莱德贡献同样对这项工作。

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Isolation of Labile Multi-protein Complexes by in vivo Controlled Cellular Cross-Linking and Immuno-magnetic Affinity Chromatography

细胞透水交联剂DSP [dithiobis（琥珀酰亚胺丙酸）]稳定短暂和不稳定的相互作用<em&gt;在体内</em&gt;，这使得他们的隔离，使用严格的蛋白质复合体的净化技术。在这里，我们目前培养的交联，由免疫蛋白复合物的分离细胞的技术。

由不稳定的多蛋白质复合物的分离在体内控制细胞的交联和免疫磁性亲和层析

The dynamic nature of cellular machineries is frequently built on transient and/or weak protein associations. These low affinity interactions preclude ...

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Biology

Isolation of Labile Multi-protein Complexes by in vivo Controlled Cellular Cross-Linking and Immuno-magnetic Affinity Chromatography

17.2K 次观看.  Emory University. 细胞透水交联剂DSP [dithiobis（琥珀酰亚胺丙酸）]稳定短暂和不稳定的相互作用在体内，这使得他们的隔离，使用严格的蛋白质复合体的净化技术。在这里，我们目前培养的交联，由免疫蛋白复合物的分离细胞的技术。

视频: Isolation of Labile Multi-protein Complexes by in vivo Controlled Cellular Cross-Linking and Immuno-magnetic Affinity Chromatography

Photo-Induced Cross-Linking of Unmodified Proteins (PICUP) Applied to Amyloidogenic Peptides

The assembly of amyloidogenic proteins into toxic oligomers is a seminal event in the pathogenesis of protein misfolding diseases, including Alzheimer's, Parkinson's, and Huntington's diseases, hereditary amyotrophic lateral sclerosis, and type 2 diabetes. Owing to the metastable nature of these protein assemblies, it is difficult to assess their oligomer size distribution quantitatively using classical methods, such as electrophoresis, chromatography, fluorescence, or dynamic light scattering. Oligomers of amyloidogenic proteins exist as metastable mixtures, in which the oligomers dissociate into monomers and associate into larger assemblies simultaneously. PICUP stabilizes oligomer populations by covalent cross-linking and when combined with fractionation methods, such as sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) or size-exclusion chromatography (SEC), PICUP provides snapshots of the oligomer size distributions that existed before cross-linking. Hence, PICUP enables visualization and quantitative analysis of metastable protein populations and can be used to monitor assembly and decipher relationships between sequence modifications and oligomerization1. Mechanistically, PICUP involves photo-oxidation of Ru2+ in a tris(bipyridyl)Ru(II) complex (RuBpy) to Ru3+ by irradiation with visible light in the presence of an electron acceptor. Ru3+ is a strong one-electron oxidizer capable of abstracting an electron from a neighboring protein molecule, generating a protein radical1,2. Radicals are unstable, highly-reactive species and therefore disappear rapidly through a variety of intra- and intermolecular reactions. A radical may utilize the high energy of an unpaired electron to react with another protein monomer forming a dimeric radical, which subsequently loses a hydrogen atom and forms a stable, covalently-linked dimer. The dimer may then react further through a similar mechanism with monomers or other dimers to form higher-order oligomers. Advantages of PICUP relative to other photo- or chemical cross-linking methods3,4 include short (&#8804;1 s) exposure to non-destructive visible light, no need for pre facto modification of the native sequence, and zero-length covalent cross-linking. In addition, PICUP enables cross-linking of proteins within wide pH and temperature ranges, including physiologic parameters. Here, we demonstrate application of PICUP to cross-linking of three amyloidogenic proteins the 40- and 42-residue amyloid &beta;-protein variants (A&beta;40 and A&beta;42), and calcitonin, and a control protein, growth-hormone releasing factor (GRF).

Photo-Induced Cross-Linking of Unmodified Proteins PICUP Applied to Amyloidogenic Peptides

Photo-induced cross-linking of unmodified proteins (PICUP) allows characterization of oligomer size distribution in metastable protein mixtures. We demonstrate application of PICUP to three representative amyloidogenic peptides the 40- and 42-residue forms of amyloid &beta;-protein, and calcitonin, and a control peptide growth-hormone releasing factor.

光致交联未修改蛋白（PICUP）适用于淀粉样肽

The assembly of amyloidogenic proteins into toxic oligomers is a seminal event in the pathogenesis of protein misfolding diseases, including Alzheimer's, ...

科研

生物学

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 ...

Visualizing Single Molecular Complexes In Vivo Using Advanced Fluorescence Microscopy

Full insight into the mechanisms of living cells can be achieved only by investigating the key processes that elicit and direct events at a cellular level. To date the shear complexity of biological systems has caused precise single-molecule experimentation to be far too demanding, instead focusing on studies of single systems using relatively crude bulk ensemble-average measurements. However, many important processes occur in the living cell at the level of just one or a few molecules; ensemble measurements generally mask the stochastic and heterogeneous nature of these events. Here, using advanced optical microscopy and analytical image analysis tools we demonstrate how to monitor proteins within a single living bacterial cell to a precision of single molecules and how we can observe dynamics within molecular complexes in functioning biological machines. The techniques are directly relevant physiologically. They are minimally-perturbative and non-invasive to the biological sample under study and are fully attuned for investigations in living material, features not readily available to other single-molecule approaches of biophysics. In addition, the biological specimens studied all produce fluorescently-tagged protein at levels which are almost identical to the unmodified cell strains ("genomic encoding"), as opposed to the more common but less ideal approach for generating significantly more protein than would occur naturally ('plasmid expression'). Thus, the actual biological samples which will be investigated are significantly closer to the natural organisms, and therefore the observations more relevant to real physiological processes.

Visualizing Single Molecular Complexes In Vivo Using Advanced Fluorescence Microscopy

Here we demonstrate the protocols for performing single-molecule fluorescence microscopy on living bacterial cells to enable functional molecular complexes to be detected, tracked and quantified.

可视化单分子络合物在体内使用高级荧光显微

Full insight into the mechanisms of living cells can be achieved only by investigating the key processes that elicit and direct events at a cellular ...

ELIME (Enzyme Linked Immuno Magnetic Electrochemical) Method for Mycotoxin Detection

Immunoassays are a valid alternative to the more expensive and time consuming quantitative HPLC or GC1, 2 methods for the screening detection of hazardous mycotoxins in food commodities. In this protocol we show how to fabricate and interrogate an electrochemical competitive Enzyme linked immunomagnetic assay based on the use of magnetic beads as solid support for the immunochemical chain3 and screen printed electrodes as sensing platform.
Our method aims to determine the total amount of HT-2 and T-2 toxins, mycotoxins belonging to the trichothecenes family and of great concern for human health4. The use of an antibody clone with a cross reactivity of 100% towards HT-2 and T-2 allows to simultaneously detect both toxins with similar sensitivity5.
The first step of our assay is the coating step where we immobilize HT2-KLH conjugate toxin on the surface of magnetic beads. After a blocking step, necessary to avoid non-specific absorptions, the addition of a monoclonal antibody allows the competition between immobilized HT-2 and free HT-2 or T-2 present in the sample or dissolved in a standard solution.
At the end of the competition step, the amount of monoclonal antibody linked to the immobilized HT-2 will be inversely proportional to the amount of toxin in the sample solution.
A secondary antibody labeled with alkaline phosphatase (AP) is used to reveal the binding between the specific antibody and the immobilized HT-2. The final measurement step is performed by dropping an aliquot of magnetic bead suspension, corresponding to a specific sample/standard solution, on the surface of a screen-printed working electrode; magnetic beads are immobilized and concentrated by means of a magnet placed precisely under the screen-printed electrode. After two minutes of incubation between magnetic beads and a substrate for AP, the enzymatic product is detected by Differential Pulse Voltammetry (DPV) using a portable instrument (PalmSens) also able to initiate automatically eight measurements within an interval of few seconds.

ELIME Enzyme Linked Immuno Magnetic Electrochemical Method for Mycotoxin Detection

A protocol to detect trichothecenes (mycotoxins of concern for human health) using a newly developed screening method based on a competitive immunochemical method and a final electrochemical detection is demonstrated.

ELIME霉菌毒素的检测方法（酶联免疫磁电化学）

Immunoassays are a valid alternative to the more expensive and time consuming quantitative HPLC or GC1, 2 methods for the screening detection of hazardous ...

In vivo Imaging of Intact Drosophila Larvae at Sub-cellular Resolution

Recent improvements in optical imaging, genetically encoded fluorophores and genetic tools allowing efficient establishment of desired transgenic animal lines have enabled biological processes to be studied in the context of a living, and in some instances even behaving, organism. In this protocol we will describe how to anesthetize intact Drosophila larvae, using the volatile anesthetic desflurane, to follow the development and plasticity of synaptic populations at sub-cellular resolution1-3. While other useful methods to anesthetize Drosophila melanogaster larvae have been previously described4,5,6,7,8, the protocol presented herein demonstrates significant improvements due to the following combined key features: (1) A very high degree of anesthetization; even the heart beat is arrested allowing for lateral resolution of up to 150 nm1, (2) a high survival rate of &gt; 90% per anesthetization cycle, permitting the recording of more than five time-points over a period of hours to days2 and (3) a high sensitivity enabling us in 2 instances to study the dynamics of proteins expressed at physiological levels. In detail, we were able to visualize the postsynaptic glutamate receptor subunit GluR-IIA expressed via the endogenous promoter1 in stable transgenic lines and the exon trap line FasII-GFP1. (4) In contrast to other methods4,7 the larvae can be imaged not only alive, but also intact (i.e. non-dissected) allowing observation to occur over a number of days1. The accompanying video details the function of individual parts of the in vivo imaging chamber2,3, the correct mounting of the larvae, the anesthetization procedure, how to re-identify specific positions within a larva and the safe removal of the larvae from the imaging chamber.

In vivo Imaging of Intact Drosophila Larvae at Sub-cellular Resolution

This protocol describes a reliable method for anesthetization and imaging of intact Drosophila melanogaster larvae. We have utilized the volatile anesthetic desflurane to allow for repetitive imaging at sub-cellular resolution and re-identification of structures for up to a few days1.

在完整的果蝇幼虫体内的亚细胞分辨率成像

Recent improvements in optical imaging, genetically encoded fluorophores and genetic tools allowing efficient establishment of desired transgenic animal ...

Isothermal Titration Calorimetry for Measuring Macromolecule-Ligand Affinity

Isothermal titration calorimetry (ITC) is a useful tool for understanding the complete thermodynamic picture of a binding reaction. In biological sciences, macromolecular interactions are essential in understanding the machinery of the cell. Experimental conditions, such as buffer and temperature, can be tailored to the particular binding system being studied. However, careful planning is needed since certain ligand and macromolecule concentration ranges are necessary to obtain useful data. Concentrations of the macromolecule and ligand need to be accurately determined for reliable results. Care also needs to be taken when preparing the samples as impurities can significantly affect the experiment. When ITC experiments, along with controls, are performed properly, useful binding information, such as the stoichiometry, affinity and enthalpy, are obtained. By running additional experiments under different buffer or temperature conditions, more detailed information can be obtained about the system. A protocol for the basic setup of an ITC experiment is given.

A general protocol for the use of isothermal titration calorimetry to monitor the binding thermodynamics for biological systems with moderate binding affinities is presented.

用于测量高分子配体亲和等温滴定量热

Isothermal titration calorimetry (ITC) is a useful tool for understanding the complete thermodynamic picture of a binding reaction.  In biological ...

Chromatin Isolation by RNA Purification (ChIRP)

Long noncoding RNAs are key regulators of chromatin states for important biological processes such as dosage compensation, imprinting, and developmental gene expression 1,2,3,4,5,6,7. The recent discovery of thousands of lncRNAs in association with specific chromatin modification complexes, such as Polycomb Repressive Complex 2 (PRC2) that mediates histone H3 lysine 27 trimethylation (H3K27me3), suggests broad roles for numerous lncRNAs in managing chromatin states in a gene-specific fashion 8,9. While some lncRNAs are thought to work in cis on neighboring genes, other lncRNAs work in trans to regulate distantly located genes. For instance, Drosophila lncRNAs roX1 and roX2 bind numerous regions on the X chromosome of male cells, and are critical for dosage compensation 10,11. However, the exact locations of their binding sites are not known at high resolution. Similarly, human lncRNA HOTAIR can affect PRC2 occupancy on hundreds of genes genome-wide 3,12,13, but how specificity is achieved is unclear. LncRNAs can also serve as modular scaffolds to recruit the assembly of multiple protein complexes. The classic trans-acting RNA scaffold is the TERC RNA that serves as the template and scaffold for the telomerase complex 14; HOTAIR can also serve as a scaffold for PRC2 and a H3K4 demethylase complex 13.
Prior studies mapping RNA occupancy at chromatin have revealed substantial insights 15,16, but only at a single gene locus at a time. The occupancy sites of most lncRNAs are not known, and the roles of lncRNAs in chromatin regulation have been mostly inferred from the indirect effects of lncRNA perturbation. Just as chromatin immunoprecipitation followed by microarray or deep sequencing (ChIP-chip or ChIP-seq, respectively) has greatly improved our understanding of protein-DNA interactions on a genomic scale, here we illustrate a recently published strategy to map long RNA occupancy genome-wide at high resolution 17. This method, Chromatin Isolation by RNA Purification (ChIRP) (Figure 1), is based on affinity capture of target lncRNA:chromatin complex by tiling antisense-oligos, which then generates a map of genomic binding sites at a resolution of several hundred bases with high sensitivity and low background. ChIRP is applicable to many lncRNAs because the design of affinity-probes is straightforward given the RNA sequence and requires no knowledge of the RNA's structure or functional domains.

Chromatin Isolation by RNA Purification ChIRP

ChIRP is a novel and rapid technique to map genomic binding sites of long noncoding RNAs (lncRNAs). The method takes advantage of the specificity of anti-sense tiling oligonucleotides to allow the enumeration of lncRNA-bound genomic sites.

RNA纯化的染色质隔离（CHIRP）

Long noncoding RNAs are key regulators of chromatin states for important biological processes such as dosage compensation, imprinting, and developmental ...

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 ...

AFM-based Mapping of the Elastic Properties of Cell Walls: at Tissue, Cellular, and Subcellular Resolutions

We describe a recently developed method to measure mechanical properties of the surfaces of plant tissues using atomic force microscopy (AFM) micro/nano-indentations, for a JPK&#160;AFM. Specifically, in this protocol we measure the apparent Young&#8217;s modulus of cell walls at subcellular resolutions across regions of up to 100 &#181;m&#160;x 100 &#181;m in floral meristems, hypocotyls, and roots. This requires careful preparation of the sample, the correct selection of micro-indenters and indentation depths. To account for cell wall properties only, measurements are performed in highly concentrated solutions of mannitol in order to plasmolyze the cells and thus remove the contribution of cell turgor pressure.
In contrast to other extant techniques, by using different indenters and indentation depths, this method allows simultaneous multiscale measurements, i.e. at subcellular resolutions and across hundreds of cells comprising a tissue. This means that it is now possible&#160;to spatially-temporally characterize the changes that take place in the mechanical properties of cell walls during development, enabling these changes to be correlated with growth and differentiation. This represents a key step to understand how coordinated microscopic cellular changes bring about macroscopic morphogenetic events.
However, several limitations remain: the method can only be used on fairly small samples (around 100 &#181;m in diameter) and only on external tissues; the method is sensitive to tissue topography; it measures only certain aspects of the tissue&#8217;s complex mechanical properties. The technique is being developed rapidly and it is likely that most of these limitations will be resolved in the near future.

We describe a method to map mechanical properties of plant tissues using an atomic force microscope (AFM). We focus on how to record mechanical changes that take place in cell walls during plant development at wide-field mesoscale, enabling these changes to be correlated with growth and morphogenesis.

细胞壁的弹性性能的原子力显微镜为基础的映射：在组织，细胞，亚细胞和决议

We describe a recently developed method to measure mechanical properties of the surfaces of plant tissues using atomic force microscopy (AFM) ...

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) maintain the turnover of all mature blood cell lineages. The coordination of the complex signals leading to specific HSC fates relies upon the interaction between HSCs and the intricate bone marrow microenvironment, which is still poorly understood[1-2].
We describe how by combining a newly developed specimen holder for stable animal positioning with multi-step confocal and two-photon in vivo imaging techniques, it is possible to obtain high-resolution 3D stacks containing HSPCs and their surrounding niches and to monitor them over time through multi-point time-lapse imaging. High definition imaging allows detecting ex vivo labeled hematopoietic stem and progenitor cells (HSPCs) residing within the bone marrow. Moreover, multi-point time-lapse 3D imaging, obtained with faster acquisition settings, provides accurate information about HSPC movement and the reciprocal interactions between HSPCs and stroma cells.
Tracking of HSPCs in relation to GFP positive osteoblastic cells is shown as an exemplary application of this method. This technique can be utilized to track any appropriately labeled hematopoietic or stromal cell of interest within the mouse calvarium bone marrow space.

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

The nature of the interactions between hematopoietic stem and progenitor cells (HSPCs) and bone marrow niches is poorly understood. Custom hardware modifications and a multi-step acquisition protocol allow the use of two-photon and confocal microscopy to image ex vivo labeled HSPCs homed within bone marrow areas, tracking interactions and movement.

在造血干细胞和祖细胞的成年小鼠颅骨骨髓体内四维跟踪

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) ...