由NIH / NIDA DA034783支持。我们感谢布赖恩·A·基兰热与多聚-PAGE技术援助。

1.组织制备<ol><li>制备4×BN-PAGE样品缓冲液（200mM双（2-羟乙基）氨基 - 三（羟甲基）甲烷（的Bis-Tris），200mM的氯化钠，40％w / v的甘油，0.004％丽春红S，pH为7.2的10毫升）。 注意:此储备溶液可以预先制成，并储存在4℃。 </li><li>稀释4倍的样品缓冲液的250μL750μL卫生署2含有1x商业蛋白酶抑制剂混合物O操作。涡和冰中冷却。 </li><li>均质化20毫克目标组织的在用干净的Dounce匀浆的30个行程1个毫升1X冰冷BN-PAGE样品缓冲液中。 注:对于这个演示实验中，目标组织是整个大鼠脑组织。均化后，样品可以用温和的去污剂如2％毛地黄皂苷处理以溶解和允许膜蛋白的电泳。 </li><li>转移匀浆到1.5mL微量离心管中，并离心以14,000×g离心30分钟以沉淀不溶细胞内容物。 AFTEř离心，倒出上清液到在冰上干净的管中。 </li><li>遵循制造商的说明书使用二辛可宁酸（BCA）或类似的蛋白定量测定法来测量上清液蛋白质浓度。 </li><li>如果匀浆样品（一个或多个）含有的洗涤剂，在水溶液中足以使匀浆溶液至0.25％考马斯添加5％考马斯蓝G-250的量。 </li></ol> 2. BN-PAGE <ol><li> 稀释25毫升20×蓝色天然（BN）电泳缓冲液的库存（1.0M的Bis-Tris，1.0M甘氨酸，pH6.8）中为475毫升含卫生署0.002％考马斯蓝，使500毫升1×电泳缓冲液的2 O。 <ol><li>如果样品含有洗涤剂，代替使250毫升含0.02％考马斯1X运行缓冲液和含有无另一个250毫升。 </li></ol></li><li>冷硬缓冲器（一个或多个），以4℃。 </li><li>清洗和组装的聚丙烯酰胺凝胶，根据制造商的说明书浇注盒。 </li><li&gt;根据标准配方7倾BN的聚丙烯酰胺凝胶（3％T堆叠层，6％T分解层），并放置好梳子。 </li><li>凝胶具有聚合后，冲洗与卫生署2 O的盒子和根据制造商说明书组装电泳装置。 </li><li> 删除以及梳子，并与1X BN-PAGE电泳缓冲液填充井。避免用缓冲液填充整个内室，直到样品被加载。 <ol><li>如果样品含有洗涤剂，填孔用含有0.02％考马斯蓝的缓冲区。 注意:在4℃下执行步骤2.7-2.9。 </li></ol></li><li>使用凝胶加载提示，吸取含有20μg蛋白质的成所需井匀浆的体积。移液器1X BN-PAGE样品缓冲液的等体积到任何未使用的孔中。 注意:使用从BCA测定法测定蛋白质浓度，以计算匀浆适当体积添加到井。 </li><li> 样品装载之后，填充用1x BN-PAGE电泳缓冲液内室。确保凝胶在缓冲液完全淹没。接着，填充用1x运行缓冲液外室由制造商所指示的电平。 <ol><li>如果样品含有洗涤剂，填用含有0.02％考马斯蓝的1X缓冲液，并用无染料1X运行缓冲液外室的内室。 </li></ol></li><li>将电极连接到电源，并在150V electrophorese将凝胶中的蛋白质直到染料带前进〜2厘米的分辨层。停止并断开电源。 </li></ol> 3.交联注意:在4℃下执行步骤3.1-3.6。 <ol><li>拆卸电泳装置中，并分离凝胶盒的玻璃板。卸下并丢弃堆叠层。 </li><li>小心切开刚好低于染料前端的底部边缘的凝胶。采取护理使这种馏分作为平直越好，然后丢弃未使用的片凝胶。这将留下〜2厘米宽，聚丙烯酰胺凝胶的水平条，其中将包含匀浆样品中的所有蛋白质。 </li><li>修剪掉沿凝胶条带的边缘的任何未使用的部分。 </li><li>小心地将条插入在一个小容器10毫升磷酸盐缓冲盐水（PBS），并轻轻地由章动30分钟以平衡混合。 </li><li>平衡之后，丢弃并用另一个10毫升替换PBS。吸移管500的μL的25mM DSP溶解于二甲基亚砜入PBS，并继续30分钟如上（步骤3.4）混合。 </li><li>倒掉DSP溶液。添加0.375的1M Tris（羟甲基）氨基甲烷盐酸盐（的Tris-HCl），pH值8.8，含有2％十二烷基硫酸钠（SDS）10毫升以猝灭未反应的DSP。继续垂头15分钟。 </li></ol> 4. SDS-PAGE <ol><li>虽然凝胶条带淬火，制备SDS-PA根据标准方法9 GE凝胶溶液。不添加聚合试剂。 </li><li> 骤冷后，返回ΒΝ凝胶条带至室温，并在带材浇注到一个新的凝胶盒。 <ol><li>要做到这一点，小心拿起凝胶，并将其放置到一个干净的凝胶盒隔板。 </li><li>东方带所以染料前端的底部最接近新磁带盒的顶部（ 即 ，在BN-PAGE行进最远的一侧应该被最接近新磁带盒的顶部;将凝胶条应从其先前被翻转方向）。参见图3。 </li><li>放置条带，使得其顶部边缘在于即使其中所述盖板的顶部边缘会（ 即 ，这将是在新的凝胶的顶部）。确保染料前端平行于所述玻璃板的水平边缘。 </li><li>推压隔板壁中的一个切下的带材的一侧上，留下上吨室他另一侧用于凝胶被倾并且要加载的蛋白质标准或梯子。 </li><li>如果凝胶条的底部边缘中包含任何锯齿或不平整区域，小心切割他们离开。如果存在的话，它们将凝胶中的气泡陷阱浇注。 </li><li>一旦凝胶条被正确地定位，笼罩间隔板盖板。轻轻推压，推出任何夹带的气泡。 </li><li>继续根据制造商说明书来组装凝胶浇注装置。 </li></ol></li><li>添加聚合试剂分离胶缓冲液，并且使用血清移液管倒在制得的凝胶盒，。填充凝胶盒至〜2厘米切除BN-PAGE凝胶带的下方，以留有余地，堆叠层。 </li><li>添加100μL丁醇在倾倒凝胶的顶部，并允许分辨层的聚合30分钟。倒掉丁醇。 </li><li> 添加聚合试剂于堆叠凝胶溶液。使用serolog移液管的iCal，倒入堆叠层以填充在凝胶盒的所有剩余空白空间。 <ol><li>倾斜凝胶盒作为层叠层倾倒，使其填充胶条下方的空间，并且气泡不会夹。 </li><li>作为层叠凝胶缓冲液填充该凝胶条下面的空的空间，逐渐将凝胶盒返回到电平的基础上。 </li><li>不断填补空白与浓缩胶缓冲切除的胶条旁边，直到它几乎溢出。 </li></ol></li><li>允许堆叠层聚合30分钟。 注意:请10X SDS-PAGE电泳缓冲液（250mM的三（羟甲基）氨基甲烷（TRIS），1.9甘氨酸，1％SDS），同时将凝胶聚合。这也可以预先制成，在室温下储存。 </li><li>稀释50毫升10X SDS-PAGE电泳缓冲液库存入450毫升卫生署2 O，使1×操作缓冲液。 </li><li>堆叠层具有聚合后，从浇注取出凝胶盒设备，冲洗与卫生署2 O，并根据制造商说明书组装电泳装置。 </li><li>用1x SDS-PAGE电泳缓冲液完全充满内腔，然后填充该外室由制造商所指示的电平。 </li><li>装入空间的分子量梯或适当蛋白质标准的切下的凝胶条带旁边。 </li><li>连接电极到电源，并且当考马斯染料跑出凝胶electrophorese在120 V.样品，停止和断开电源。 </li><li>分析使用电印迹和蛋白质检测的标准方法通过抗体结合10的凝胶。 </li></ol>

<ol>
	<li>Alberts, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+cell+as+a+collection+of+protein+machines:+preparing+the+next+generation+of+molecular+biologists.">The cell as a collection of protein machines: preparing the next generation of molecular biologists.</a> Cell. 92 (3), 291-294 (1998).</li><li>Pawson, T., Nash, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein-protein+interactions+define+specificity+in+signal+transduction.">Protein-protein interactions define specificity in signal transduction.</a> Genes Dev. 14 (9), 1027-1047 (2000).</li><li>Rual, J. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Towards+a+proteome-scale+map+of+the+human+protein-protein+interaction+network.">Towards a proteome-scale map of the human protein-protein interaction network.</a> Nature. 437 (7062), 1173-1178 (2005).</li><li>Phizicky, E. M., Fields, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein-protein+interactions:+methods+for+detection+and+analysis.">Protein-protein interactions: methods for detection and analysis.</a> Microbiol Rev. 59 (1), 94-123 (1995).</li><li>Ho, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Systematic+identification+of+protein+complexes+in+Saccharomyces+cerevisiae+by+mass+spectrometry.">Systematic identification of protein complexes in Saccharomyces cerevisiae by mass spectrometry.</a> Nature. 415 (6868), 180-183 (2002).</li><li>Krogan, N. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Global+landscape+of+protein+complexes+in+the+yeast+Saccharomyces+cerevisiae.">Global landscape of protein complexes in the yeast Saccharomyces cerevisiae.</a> Nature. 440 (7084), 637-643 (2006).</li><li>Wittig, I., Braun, H. P., Schagger, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blue+native+PAGE.">Blue native PAGE.</a> Nat Protoc. 1 (1), 418-428 (2006).</li><li>Schagger, H., Cramer, W. A., von Jagow, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+molecular+masses+and+oligomeric+states+of+protein+complexes+by+blue+native+electrophoresis+and+isolation+of+membrane+protein+complexes+by+two-dimensional+native+electrophoresis.">Analysis of molecular masses and oligomeric states of protein complexes by blue native electrophoresis and isolation of membrane protein complexes by two-dimensional native electrophoresis.</a> Anal Biochem. 217 (2), 220-230 (1994).</li><li>Laemmli, U. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cleavage+of+structural+proteins+during+the+assembly+of+the+head+of+bacteriophage+T4.">Cleavage of structural proteins during the assembly of the head of bacteriophage T4.</a> Nature. 227 (5259), 680-685 (1970).</li><li>Beisiegel, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+Blotting.">Protein Blotting.</a> Electrophoresis. 7 (1), 1-18 (1986).</li><li>Killinger, B. A., Moszczynska, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+alpha-Synuclein+Multimer+Stoichiometry+in+Complex+Biological+Samples+by+Electrophoresis.">Characterization of alpha-Synuclein Multimer Stoichiometry in Complex Biological Samples by Electrophoresis.</a> Anal Chem. 88 (7), 4071-4084 (2016).</li><li>Newman, A. J., Selkoe, D., Dettmer, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+method+for+quantitative+immunoblotting+of+endogenous+alpha-synuclein.">A new method for quantitative immunoblotting of endogenous alpha-synuclein.</a> PLoS One. 8 (11), e81314 (2013).</li><li>Wong, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Chemistry of protein conjugation and cross-linking. , (1991).</li><li>Seddon, A. M., Curnow, P., Booth, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Membrane+proteins,+lipids+and+detergents:+not+just+a+soap+opera.">Membrane proteins, lipids and detergents: not just a soap opera.</a> Biochim Biophys Acta. 1666 (1-2), 105-117 (2004).</li><li>Tanford, C., Reynolds, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+membrane+proteins+in+detergent+solutions.">Characterization of membrane proteins in detergent solutions.</a> Biochim Biophys Acta. 457 (2), 133-170 (1976).</li><li>Peters, K., Richards, F. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemical+cross-linking:+reagents+and+problems+in+studies+of+membrane+structure.">Chemical cross-linking: reagents and problems in studies of membrane structure.</a> Annu Rev Biochem. 46, 523-551 (1977).</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 (1), 243-261 (1976).</li><li>Sun, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Crystal+structure+of+mitochondrial+respiratory+membrane+protein+complex+II.">Crystal structure of mitochondrial respiratory membrane protein complex II.</a> Cell. 121 (7), 1043-1057 (2005).</li><li>Wittig, I., Schagger, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Native+electrophoretic+techniques+to+identify+protein-protein+interactions.">Native electrophoretic techniques to identify protein-protein interactions.</a> Proteomics. 9 (23), 5214-5223 (2009).</li></ol>

作者什么都没有透露。

蛋白质 - 蛋白质相互作用是每个任务万物开展重要。正因为如此，他们是严格审查和研究的课题。多聚-PAGE是用于捕获，分离，和分析广泛的蛋白复合物的新方法。之前我们已经证实其适用于研究疾病相关蛋白α突触核蛋白11 oligimerization。然而，它是可扩展到许多蛋白复合物，如在图2证实。当与学习蛋白复合物的其他方法，多聚-PAGE是因为它的简单和简洁的是有利的。从组织样本的时间完全分离SDS凝胶为约8小时。由于检测可以通过典型的免疫印迹来完成，该协议可以在未修饰的组织裂解液中进行，以检测限比用下拉实验相关联的那些低得多。此外，装备精良的大多数分子生物学拉博斋戒者将能够以很少的前期投资进行这项技术。如稍后讨论的，与多聚体PAGE相关联的大多数挑战来自于对所研究的每种蛋白质复合物的方法进行故障排除和优化。 尽管多分子生物学家可以访问多聚片段，但其成功取决于研究者的技能和经验。重要的是，必须小心地将BN凝胶条切割并重新投入新的SDS凝胶。重要的是使凝胶条定向，使电泳使蛋白质以平行条带迁移到SDS凝胶中。如果凝胶条弯曲，蛋白质带将相互迁移，数据将丢失。特别是对于大复合物，将凝胶条旋转到SDS-PAGE凝胶之前也是同样重要的。这产生较大的络合物，其可能不会迁移到非常远的BN-PAGE凝胶，更多的时间被拉入分辨层的SDS-PAGE凝胶的河重要的是，造成交联的更改蛋白尺寸和物理特性也应分析期间考虑。例如，一些低分子量蛋白质，如α突触核蛋白，不能很好地保持在PVDF膜上，但它们的交联的低聚物是12。这可以混淆对比分析，而且必须加以考虑。 当由多聚-PAGE首次分离的复合物，它来验证方法是重要的。我们推荐三个验证/故障诊断测试。首先，确保感兴趣的肽迁移入BN凝胶由以下多聚-PAGE协议直到凝胶条的切除，然后停止，条带印迹到PVDF膜上，并且探测所关心的子单元。如果不存在信号，该复合物很可能未迁移进入BN凝胶，这意味着它应更加多孔的，或样品应被允许进一步迁移到RESOLVING层。的信号的存在确认，至少未结合的亚基迁移到凝胶。在这个阶段，这将是难以确认的全复合物的存在。如果BN凝胶条带被检测到亚基的证据，移动到所述第二测试。没有交联步骤进行多聚-PAGE，然后印迹到PVDF膜上并探针用于亚基。子单元应​​SDS存在变性和正常迁移到SDS凝胶。如果这种情况不会发生，有可能有些问题研究者的技术。的BN凝胶条带可能已被切断不佳，留下一些蛋白质的后面，或SDS凝胶可能已被错误地铸造。最后，执行多聚-PAGE与包含交联剂裂解步骤，然后印迹和探针，如在图1中所讨论。这证实了复合物的聚集是由于除了DSP的，而不是多聚-PAGE协议的一些其他部分的意外的结果。裂解应导致减少D-强度骨料带和成像时的增加的强度亚基单体频带。如果没有聚合带由正常多聚-PAGE观察，但在强度单体带增大时DSP被裂解，所述复合物可能已经做了它对SDS凝胶，但未能迁移到分辨层。 在其目前状态，多聚-PAGE是适用于许多蛋白质复合物，但不是一个尺寸适合所有协议。它可能需要修改为每个感兴趣的复杂。一个常见的问题是体现在图2:捕获复合物经常是如此之大，它们在SDS-PAGE迁移几乎没有。这可以通过改变SDS凝胶的孔隙率或更长的时间段进行电泳来管理。额外的复杂性是在凝胶上多于两个条带的偶然存在。与交联剂反应不完全可能会导致中间分子量条带13的分布。这可以使DIFficult以确定多个频带是否具有代表性生理上重要的寡聚中间体，或部分交联的简单意想不到的后果的。对于必须分析之前溶解膜蛋白，它也是重要的考虑因素是洗涤剂的选择对蛋白复合物的行为的影响。用来溶解膜蛋白的洗涤剂影响他们的构象，与关联结合配偶体，和功能14,15。洗涤剂选择也影响的交联16的功效。因此，很可能的是，理想的增溶条件将随所研究的复杂变化。 多聚-PAGE偶尔不能捕获的复合物，如在图2中琥珀酸脱氢酶的SDHA印迹示出的是124 kDa的线粒体复合物II，其足够小，但应该容易地移动到凝胶的一部分。这是不是检测表明，多聚-PAGE无法捕捉到它。此类情况下有很多可能的解释。大多数交联试剂包含两个反应性端部，其形成与氨基酸侧链共价键。效率与交联试剂捕获的蛋白复合物是依赖于它们的可访问到的反应性残基。在DSP的情况下，交联是通过暴露于溶剂的伯和仲脂族氨基，通常赖氨酸17的ε氨基的酰化来完成。赖氨酸残基的蛋白质表面上通常发现，但是它们在疏水性中心偶尔螯合降低的交联13的效率。复合体II的亚基都埋在线粒体膜，甚至与毛地黄皂苷增溶18后可能仍然无法访问DSP，。非常大的，密集的包装物，如淀粉样蛋白oligomeRS，也可以限制表面赖氨酸残基的可用性以交联试剂。其结果是，这些类型的蛋白质组件可能不与多聚-PAGE兼容。它也有可能是考马斯蓝，其结合的疏水区和阳离子残基，平衡在PBS中，并阻止交联在某些情况下19后徘徊。这可能会减少或消除多聚-PAGE检测影响低聚物。因此，该方法不用于表征所有蛋白质关联的通用试验，并且其中在定量期望不应该被考虑。然而，多聚-PAGE是用于分析的蛋白质复合物的廉价的，相对迅速的工具，并且可以与其他装置建立被考虑。

<html>正常细胞功能是依赖于蛋白质-蛋白质相互作用1,2。其结果是，人类疾病通常是由在不同的蛋白质复合物3的组件和行为的扰动标记。表征这种相互作用的能力因此，至关重要。检测这些相互作用电流的装置需要的靶蛋白，通常随后通过相互作用配偶的下拉的纯化。常规纯化通过差速离心，沉淀和/或色谱法4来实现的。这些方法是耗时的，必须改变用于每个靶蛋白，并经常导致低的产率。现代纯化方法涉及肽标签融合到靶蛋白，随后通过免疫沉淀或萃取上装载有结合于捕获分子5珠粒柱，"&gt; 6，虽然这是可扩展到许多蛋白质，这需要目标的序列修饰，潜在地导致在亲和力差异，它们通常的结合配偶体的构建体。一些蛋白质-蛋白质相互作用的微妙特性意味着这种方法可能不适用许多方案。另外，用于映射蛋白质 - 蛋白质相互作用的下拉测定法可以不捕获准确蜂窝图片，由于受限制的自由度和诱饵蛋白的非天然的水平。 理想情况下，蛋白复合物能够在他们的原生状态进行检测，而不需要净化或下拉。蓝色非变性聚丙烯酰胺凝胶电泳（BN-PAGE）被开发作为变性少替代十二烷基硫酸钠聚丙烯酰胺凝胶电泳（SDS-PAGE），并允许某些蛋白质和复合物从生物样品7的分离。然而，在BN-PAGE蛋白质分离的基础上LARGE数的变量，包括大小，电荷，三维结构，和关联与其他分子。这些因素的相互作用通常导致蛋白质的共分离，聚集体形成，差的蛋白质条带的分辨率。二维原生聚丙烯酰胺凝胶电泳解决了一些，但不是全部，这些问题8。 为了避开与天然分离相关的并发症，一些作者使用胺反应性的交联试剂，如二硫代双（琥珀酰亚胺丙酸酯）（DSP），以捕获蛋白复合物在组织裂解物4。然后将这些处理过的溶胞产物可变性，并通过SDS-PAGE分离，同时保持蛋白质复合物的天然大小和化妆。然而，由于交联试剂反应基于一个分子到另一个的接近度，以及在组织裂解液的蛋白质具有许多自由度，并且可以随机相互作用，非特异性背景交叉-linking可以是高的，尤其是浓缩的样品英寸这可能会导致难以解释结果。 在这里，我们证明了使用混合BN-PAGE / SDS-PAGE的方法，被称为多聚聚丙烯酰胺凝胶电泳（多聚-PAGE），以分离和复杂混合物中检测蛋白组件。最初，细胞裂解物通过BN-PAGE悬浮于聚丙烯酰胺凝胶。含溶胞产物 - 凝胶，然后用交联试剂DSP反应。伪固定，并略微在凝胶上分离，蛋白可能非特异性反应少得多，这意味着背景交联剂反应性降低。交联后，将凝胶条带切下并通过SDS-PAGE分离。将所得的凝胶然后可以通过典型地与聚丙烯酰胺凝胶电泳相关的任何手段进行分析。这种方法允许在未修饰的组织裂解物天然蛋白质复合物的分离和检测，而无需额外的纯化或上拉陶氏ñ。

<table border="0" cellpadding="0" cellspacing="0" width="817">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:336px;">Chemicals</td>
			<td style="width:195px;"></td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:336px;">&#949;-Aminocaproic acid</td>
			<td style="width:195px;">Sigma</td>
			<td style="width:119px;">A2504</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:336px;">Acrylamide</td>
			<td style="width:195px;">Acros Organics</td>
			<td style="width:119px;">164855000</td>
			<td>Toxic.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:336px;">Acrylamide/bisacrilamide 37.5:1 (40%T stock solution)</td>
			<td style="width:195px;">BioRad</td>
			<td style="width:119px;">161-0148</td>
			<td>Toxic.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:336px;">Ammonium persulfate</td>
			<td style="width:195px;">Sigma</td>
			<td style="width:119px;">A3678</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:336px;">Anti rabbit IgG-HRP from goat</td>
			<td style="width:195px;">Santa Cruz Biotechnology</td>
			<td style="width:119px;">sc-2004</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:336px;">Bicinchoninic acid assay kit</td>
			<td style="width:195px;">Thermofisher</td>
			<td style="width:119px;">23225</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:336px;">Bis-tris</td>
			<td style="width:195px;">Sigma</td>
			<td style="width:119px;">B9754</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:336px;">Bovine serum albumin</td>
			<td style="width:195px;">Sigma</td>
			<td style="width:119px;">A9647</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:336px;">Butanol</td>
			<td style="width:195px;">Fisher Scientific</td>
			<td style="width:119px;">A399-1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:336px;">Chemiluminescence substrate kit</td>
			<td style="width:195px;">ThermoFisher</td>
			<td style="width:119px;">24078</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:336px;">Coomassie blue G-250</td>
			<td style="width:195px;">Sigma-Aldrich</td>
			<td style="width:119px;">B0770</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:336px;">Digitonin</td>
			<td style="width:195px;">Sigma</td>
			<td style="width:119px;">D141</td>
			<td>Toxic.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:336px;">Dimethyl sulfoxide</td>
			<td style="width:195px;">Fisher Scientific</td>
			<td style="width:119px;">D128-1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:336px;">Dithiobis(succinimidylpropionate)</td>
			<td style="width:195px;">Thermo Scientific</td>
			<td style="width:119px;">22585</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:336px;">Dry nonfat milk</td>
			<td style="width:195px;">LabScientific</td>
			<td style="width:119px;">M0841</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:336px;">Glycerol</td>
			<td style="width:195px;">Sigma</td>
			<td style="width:119px;">G9012</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:336px;">Glycine</td>
			<td style="width:195px;">Fisher Scientific</td>
			<td style="width:119px;">BP381-5</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:336px;">Halt Protease Inhibitor Cocktail</td>
			<td style="width:195px;">Thermofisher</td>
			<td>78430</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:336px;">Hydrochloric acid</td>
			<td style="width:195px;">Fisher Scientific</td>
			<td style="width:119px;">A144SI-212</td>
			<td>For titration. Caustic.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:336px;">Methanol</td>
			<td style="width:195px;">Fisher Scientific</td>
			<td style="width:119px;">A412-4</td>
			<td>For PVDF membrane activation. Toxic.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:336px;">Monoclonal anti &#945;-synuclein IgG from rabbit</td>
			<td style="width:195px;">Santa Cruz Biotechnology</td>
			<td style="width:119px;">sc-7011-R</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:336px;">N,N,N&#39;,N&#39;-tetramethylethylenediamine</td>
			<td style="width:195px;">Sigma</td>
			<td style="width:119px;">T9281</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:336px;">N,N&#39;-methylenebisacrylamide</td>
			<td style="width:195px;">Acros Organics</td>
			<td style="width:119px;">16479</td>
			<td>Toxic.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:336px;">NP40</td>
			<td style="width:195px;">Boston Bioproducts</td>
			<td style="width:119px;">P-872</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:336px;">Polysorbate 20 (tween-20)</td>
			<td style="width:195px;">Fisher Scientific</td>
			<td style="width:119px;">BP337-500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:336px;">Polyvinylidene fluoride transfer membranes</td>
			<td style="width:195px;">Thermo Scientific</td>
			<td style="width:119px;">88518</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:336px;">Ponceau S</td>
			<td style="width:195px;">Sigma</td>
			<td style="width:119px;">P3504</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:336px;">Potassium chloride</td>
			<td style="width:195px;">Fisher Scientific</td>
			<td style="width:119px;">P217-3</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:336px;">Potassium phosphate monobasic</td>
			<td style="width:195px;">Sigma</td>
			<td style="width:119px;">P9791</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:336px;">Protease inhibitor cocktail</td>
			<td style="width:195px;">Thermo Scientific</td>
			<td style="width:119px;">88265</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:336px;">SDS solution (10% w/v)</td>
			<td style="width:195px;">BioRad</td>
			<td style="width:119px;">161-0416</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:336px;">Sodium chloride</td>
			<td style="width:195px;">Fisher Scientific</td>
			<td style="width:119px;">BP358-212</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:336px;">Sodium dodecyl sulfate</td>
			<td style="width:195px;">Sigma</td>
			<td style="width:119px;">L37771</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:336px;">Sodium phosphate monobasic</td>
			<td style="width:195px;">Fisher Scientific</td>
			<td style="width:119px;">BP329-1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:336px;">Tricine</td>
			<td style="width:195px;">Sigma</td>
			<td style="width:119px;">T0377</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:336px;">Tris base</td>
			<td style="width:195px;">Fisher Scientific</td>
			<td style="width:119px;">BP152-500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:336px;">Tris-HCl (0.5 M buffer, pH 6.8)</td>
			<td style="width:195px;">BioRad</td>
			<td style="width:119px;">161-0799</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:336px;">Tris-HCl (1.5 M buffer, pH 8.8)</td>
			<td style="width:195px;">BioRad</td>
			<td style="width:119px;">161-0798</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:336px;">Name</td>
			<td style="width:195px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Instruments</td>
			<td style="width:195px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:336px;">GE Imagequant LAS 4000</td>
			<td style="width:195px;">GE Healthcare</td>
			<td style="width:119px;">28-9558-10</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:336px;">ImageJ software</td>
			<td style="width:195px;">NIH</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:336px;">Synergy H1 microplate reader</td>
			<td style="width:195px;">BioTek</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:336px;">Gel Former + Stand</td>
			<td style="width:195px;">Biorad</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:336px;">Microfuge 22R centrifuge</td>
			<td style="width:195px;">Beckman Coulter</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:336px;">2 mL dounce homogenizer</td>
			<td style="width:195px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:336px;">Vortex mixer</td>
			<td style="width:195px;">Fisher Scientific</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:336px;">Ultrasonic tissue homogenizer</td>
			<td style="width:195px;">Fisher Scientific</td>
			<td style="width:119px;">FB120220</td>
			<td></td>
		</tr>
	</tbody>
</table>

multimer-page: a method for capturing and resolving protein complexes in biological samples

有许多用于净化和研究单个蛋白质和肽发达的方法。然而，大多数的细胞功能是由相互作用的蛋白复合物的网络，这往往是难以调查，因为它们的结合是非共价的，并通过纯化技术容易扰动进行。这项工作描述了稳定剂和使用二维聚丙烯酰胺凝胶电泳从未经修饰的组织分离天然蛋白质复合物的方法。组织裂解液加载到非变性蓝色天然聚丙烯酰胺凝胶，然后施加电电流，直到蛋白质迁移的短距离到凝胶。然后将含有已迁移蛋白质凝胶条带切出，并与该胺反应性交联剂二硫代双（丙酸琥珀酰亚胺酯），其共价稳定的蛋白复合物一起温育。然后将含有交联复合物的凝胶条带浇注成十二烷基硫酸钠聚丙烯酰胺凝胶，和叔他配合是完全分开的。该方法依赖于技术和最熟悉的分子生物学家的材料，这意味着它是价格低廉，简单易学。虽然在其充分分离非常大的复合能力是有限的，并没有受到普遍成功，该方法能够捕捉各种充分研究的复合物，并有可能适用于许多感兴趣的系统。

在这个演示实验，多聚-PAGE是在整个大鼠脑裂解液进行。将所得的分离的蛋白质印迹到聚偏二氟乙烯（PVDF）膜上，然后用针对已知能形成复合物的蛋白质的抗体进行探测。 图1示出了通过两种方式的协议的验证。首先，我们表明，交联的蛋白是可裂解通过加入还原剂的，这意味着所观察到的较高分子量的物质通过交联试剂形成，并且不是由于协议（ 图1A）的任何其它部件。第二，我们表明，交联是除了去污剂（ 图1B）的敏感，这意味着交联是特定于复合的蛋白质和，由于关闭凝胶上接近随机的反应性最小化。 图2表明，该方法未能捕捉RESPiratory复合物II，但除此之外，具有用于捕获可溶性和膜结合的复合物广泛的适用性。 <img alt="图1" src="/files/ftp_upload/55341/55341fig1.jpg" /> 图1:多聚-PAGE方法的验证。 （A）通过在凝胶与DSP交联蛋白复合物的捕获是通过二硫苏糖醇（DTT）裂解敏感。无（A1）或与整个大鼠脑裂解物进行多聚-PAGE（A2）的5mM二硫苏糖醇包括在TRIS / SDS溶液急冷。将凝胶电吸印到PVDF膜上，然后探测驱动蛋白重链。高分子量带在两个膜上观察到的，可能对应于所有或驱动蛋白马达蛋白复合物的一部分。有在126 kDa的上DTT处理的印迹，驱动蛋白重链肽的分子量发生的附加频带。二硫苏糖醇是一种还原剂，并能够反向CR通过切割存在于DSP二硫键OSS联。驱动蛋白聚集体是由DTT裂解因此敏感。 （B）蛋白质复合物的捕获是加成变性洗涤剂敏感。多聚-PAGE被如文中所描述的，除了SDS（左）或Triton X-100（右）加入到裂解物样品，然后在冰上温育的增加量（0至2％）对整个大鼠脑裂解物进行用于装载第一凝胶前30分钟。最终的凝胶印迹到PVDF膜上，并探查α突触核蛋白。提高分子量的至少三个频带观察到，对应于α-突触核蛋白单体，四聚体，八聚体及分别。低聚频带的信号强度降低与洗涤剂浓度的增加，这表明交联效力取决于天然蛋白质构象的。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55341/55341fig1large.jpg" target="_blank">请点击此处为vi</a>EW此图的放大版本。 <img alt="图2" src="/files/ftp_upload/55341/55341fig2.jpg" /> 图2:使用多聚-PAGE蛋白质复合物的捕获的示范。 （A）可溶性的和膜结合蛋白复合物由（A）整体大鼠脑裂解物或在4℃下用2％洋地黄皂苷温育1个小时，通过如文中所述进行多聚-PAGE（B）裂解物捕获。然后，将凝胶吸干并探讨各种复合物成分的PVDF膜。高分子量物质是在探查乙酰化微管蛋白，这是众所周知的稳定微管的膜观察到。动力蛋白和驱动蛋白是多亚基马达蛋白复合物与分别为1.5 MDA和380 kDa的分子量。涂抹这些复合物成分的膜显示出高分子量的物质。此外，多聚-PAGE成功Çaptured的HSP90二聚体。 α-突触核蛋白形成四聚体八聚体和，以及神经毒性高分子量的低聚物。八聚体和更高的重量物种的条纹上看到的印迹膜。 VDAC二聚化，也观察到。上印迹线粒体复合物I和IV的成分的膜中检测到的高分子量物质。然而，膜印迹为SDHA，复合体II的一员，不表现出任何合适的高分子量条带。 AcetTUB:乙酰化的α微管蛋白; βTUB:β微管蛋白;动力蛋白:动力蛋白重链; HSP90:热休克蛋白90;驱动蛋白:驱动蛋白重链; NDUFS3:线粒体复合物的铁 - 硫中心3 I; VDAC:孔蛋白; SDHA:线粒体复合物II的琥珀酸脱氢酶; COXIV:线粒体复合物IV的细胞色素C氧化。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55341/55341fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 图3:一般多聚-PAGE流程图。 （A）使用非变性方法，如在BN-PAGE样品缓冲液均化制备的组织裂解液。 （B）吸管裂解物进BN-PAGE凝胶，然后执行在BN-PAGE电泳缓冲液电泳（150伏）。允许样品至2厘米迁移直至染料前沿已经迁移〜入分离胶。 （C）取出堆叠层和分辨层的未使用部分。 （D）淹没在PBS中凝胶带，并振摇30分钟平衡。 （E）取出PBS，并重新淹没在PBS含有2.5mM DSP的凝胶条。摇动30分钟。 （F）取下DSP溶液，然后重新淹没在375毫摩尔Tris含有2％SDS凝胶带，并摇动30分钟。存在于凝胶条络合物现在由合同研究组织稳定S-链接。 （G）旋转凝胶条180°并浇注到一个新的SDS-PAGE凝胶。包括堆叠和​​解决层。 （H）解决通过电泳（120伏）的样品。 （I）中除去凝胶带和层叠层，然后分析分辨层是必要的。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55341/55341fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about Multimer-PAGE: A Method for Capturing and Resolving Protein Complexes in Biological Samples at JoVE.com

Multimer-PAGE: A Method for Capturing and Resolving Protein Complexes in Biological Samples

一种用于稳定和使用胺反应性蛋白交联剂偶联到一种新颖的二维聚丙烯酰胺凝胶电泳分离未修饰的组织裂解物天然蛋白质复合物的方法（PAGE）系统被呈现。

多聚-PAGE:生物样品中的用于拍摄方法和解决蛋白复合体

There are many well-developed methods for purifying and studying single proteins and peptides. However, most cellular functions are carried out by ...

multimer-page-method-for-capturing-resolving-protein-complexes

Nanyang Technological University

Research

JoVE Journal

Biochemistry

11.3K 次观看.  Michigan State University. 一种用于稳定和使用胺反应性蛋白交联剂偶联到一种新颖的二维聚丙烯酰胺凝胶电泳分离未修饰的组织裂解物天然蛋白质复合物的方法（PAGE）系统被呈现。 

视频: Multimer-PAGE: A Method for Capturing and Resolving Protein Complexes in Biological Samples

Sheathless Capillary Electrophoresis&#8211;Mass Spectrometry for Metabolic Profiling of Biological Samples

In metabolomics, a wide range of analytical techniques is used for the global profiling of (endogenous) metabolites in complex samples. In this paper, a protocol is presented for the analysis of anionic and cationic metabolites in biological samples by capillary electrophoresis&#8211;mass spectrometry (CE-MS). CE is well-suited for the analysis of highly polar and charged metabolites as compounds are separated on the basis of their charge-to-size ratio. A recently developed sheathless interfacing design, i.e., a porous tip interface, is used for coupling CE to electrospray ionization (ESI) MS. This interfacing approach allows the effective use of the intrinsically low-flow property of CE in combination with MS, resulting in nanomolar detection limits for a broad range of polar metabolite classes. The protocol presented here is based on employing a bare fused-silica capillary with a porous tip emitter at low-pH separation conditions for the analysis of a broad array of metabolite classes in biological samples. It is demonstrated that the same sheathless CE-MS method can be used for the profiling of cationic metabolites, including amino acids, nucleosides and small peptides, or anionic metabolites, including sugar phosphates, nucleotides and organic acids, by only switching the MS detection and separation voltage polarity. Highly information-rich metabolic profiles in various biological samples, such as urine, cerebrospinal fluid and extracts of the glioblastoma cell line, can be obtained by this protocol in less than 1 hr of CE-MS analysis.

A protocol for metabolic profiling of biological samples by capillary electrophoresis&#8211;mass spectrometry using a sheathless porous tip interface design is presented.

鞘毛细管电泳 - 质谱联用生物样品的代谢谱

In metabolomics, a wide range of analytical techniques is used for the global profiling of (endogenous) metabolites in complex samples. In this paper, a ...

科研

生物化学

Method for Identifying Small Molecule Inhibitors of the Protein-protein Interaction Between HCN1 and TRIP8b

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are expressed ubiquitously throughout the brain, where they function to regulate the excitability of neurons. The subcellular distribution of these channels in pyramidal neurons of hippocampal area CA1 is regulated by tetratricopeptide repeat-containing Rab8b interacting protein (TRIP8b), an auxiliary subunit. Genetic knockout of HCN pore forming subunits or TRIP8b, both lead to an increase in antidepressant-like behavior, suggesting that limiting the function of HCN channels may be useful as a treatment for Major Depressive Disorder (MDD). Despite significant therapeutic interest, HCN channels are also expressed in the heart, where they regulate rhythmicity. To circumvent off-target issues associated with blocking cardiac HCN channels, our lab has recently proposed targeting the protein-protein interaction between HCN and TRIP8b in order to specifically disrupt HCN channel function in the brain. TRIP8b binds to HCN pore forming subunits at two distinct interaction sites, although here the focus is on the interaction between the tetratricopeptide repeat (TPR) domains of TRIP8b and the C terminal tail of HCN1. In this protocol, an expanded description of a method for purifying TRIP8b and executing a high throughput screen to identify small molecule inhibitors of the interaction between HCN and TRIP8b, is described. The method for high throughput screening utilizes a Fluorescence Polarization (FP) -based assay to monitor the binding of a large TRIP8b fragment to a fluorophore-tagged eleven amino acid peptide corresponding to the HCN1 C terminal tail. This method allows &#39;hit&#39; compounds to be identified based on the change in the polarization of emitted light. Validation assays are then performed to ensure that &#39;hit&#39; compounds are not artifactual.

The interaction between HCN channels and their auxiliary subunit has been identified as a therapeutic target in Major Depressive Disorder. Here, a fluorescence polarization-based method for identifying small molecule inhibitors of this protein-protein interaction, is presented.

方法确定HCN1和TRIP8b之间蛋白 - 蛋白相互作用的小分子抑制剂

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are expressed ubiquitously throughout the brain, where they function to regulate the ...

Time-resolved ElectroSpray Ionization Hydrogen-deuterium Exchange Mass Spectrometry for Studying Protein Structure and Dynamics

Intrinsically disordered proteins (IDPs) have long been a challenge to structural biologists due to their lack of stable secondary structure elements. Hydrogen-Deuterium Exchange (HDX) measured at rapid time scales is uniquely suited to detect structures and hydrogen bonding networks that are briefly populated, allowing for the characterization of transient conformers in native ensembles. Coupling of HDX to mass spectrometry offers several key advantages, including high sensitivity, low sample consumption and no restriction on protein size. This technique has advanced greatly in the last several decades, including the ability to monitor HDX labeling times on the millisecond time scale. In addition, by incorporating the HDX workflow onto a microfluidic platform housing an acidic protease microreactor, we are able to localize dynamic properties at the peptide level. In this study, Time-Resolved ElectroSpray Ionization Mass Spectrometry (TRESI-MS) coupled to HDX was used to provide a detailed picture of residual structure in the tau protein, as well as the conformational shifts induced upon hyperphosphorylation.

Conformational flexibility plays a critical role in protein function. Herein, we describe the use of time-resolved electrospray ionization mass spectrometry coupled to hydrogen-deuterium exchange for probing the rapid structural changes that drive function in ordered and disordered proteins.

时间分辨电喷雾电离氢氘交换质谱用于研究蛋白质结构与动态

Intrinsically disordered proteins (IDPs) have long been a challenge to structural biologists due to their lack of stable secondary structure elements. ...

Resolving Affinity Purified Protein Complexes by Blue Native PAGE and Protein Correlation Profiling

Most proteins act in association with others; hence, it is crucial to characterize these functional units in order to fully understand biological processes. Affinity purification coupled to mass spectrometry (AP-MS) has become the method of choice for identifying protein-protein interactions. However, conventional AP-MS studies provide information on protein interactions, but the organizational information is lost. To address this issue, we developed a strategy to unravel the distinct functional assemblies a protein might be involved in, by resolving affinity-purified protein complexes prior to their characterization by mass spectrometry. Protein complexes isolated through affinity purification of a bait protein using an epitope tag and competitive elution are separated through blue native electrophoresis. Comparison of protein migration profiles through correlation profiling using quantitative mass spectrometry allows assignment of interacting proteins to distinct molecular entities. This method is able to resolve protein complexes of close molecular weights that might not be resolved by traditional chromatographic techniques such as gel filtration. With little more work than conventional AP-geLC-MS/MS, we demonstrate this strategy may in many cases be adequate for obtaining protein complex topological information concomitantly to identifying protein interactions.

Here we present protocols for affinity purification of protein complexes and their separation by blue native PAGE, followed by protein correlation profiling using label free quantitative mass spectrometry. This method is useful to resolve interactomes into distinct protein complexes.

解决由蓝变性PAGE和蛋白质相关剖析亲和纯化的蛋白复合物

Most proteins act in association with others; hence, it is crucial to characterize these functional units in order to fully understand biological ...

Detection and Visualization of DNA Damage-induced Protein Complexes in Suspension Cell Cultures Using the Proximity Ligation Assay

The DNA damage response orchestrates the repair of DNA lesions that occur spontaneously, are caused by genotoxic stress, or appear in the context of programmed DNA breaks in lymphocytes. The Ataxia-Telangiectasia Mutated kinase (ATM), ATM- and Rad3-Related kinase (ATR) and the catalytic subunit of DNA-dependent Protein Kinase (DNA-PKcs) are among the first that are activated upon induction of DNA damage, and are central regulators of a network that controls DNA repair, apoptosis and cell survival. As part of a tumor-suppressive pathway, ATM and ATR activate p53 through phosphorylation, thereby regulating the transcriptional activity of p53. DNA damage also results in the formation of so-called ionizing radiation-induced foci (IRIF) that represent complexes of DNA damage sensor and repair proteins that accumulate at the sites of DNA damage, which are visualized by fluorescence microscopy. Co-localization of proteins in IRIFs, however, does not necessarily imply direct protein-protein interactions, as the resolution of fluorescence microscopy is limited. 
In situ Proximity Ligation Assay (PLA) is a novel technique that allows the direct visualization of protein-protein interactions in cells and tissues with unprecedented specificity and sensitivity. This technique is based on the spatial proximity of specific antibodies binding to the proteins of interest. When the interrogated proteins are within ~40 nm an amplification reaction is triggered by oligonucleotides that are conjugated to the antibodies, and the amplification product is visualized by fluorescent labeling, yielding a signal that corresponds to the subcellular location of the interacting proteins. Using the established functional interaction between ATM and p53 as an example, it is demonstrated here how PLA can be used in suspension cell cultures to study the direct interactions between proteins that are integral parts of the DNA damage response.

Here, it is demonstrated how the in situ Proximity Ligation Assay (PLA) can be used to detect and visualize the direct protein-protein interactions between ATM and p53 in suspension cell cultures exposed to genotoxic stress.

检测和可视化DNA损伤诱导的蛋白质复合物悬浮细胞培养使用邻近连接测定

The DNA damage response orchestrates the repair of DNA lesions that occur spontaneously, are caused by genotoxic stress, or appear in the context of ...

Horizontal Gel Electrophoresis for Enhanced Detection of Protein-RNA Complexes

Native polyacrylamide gel electrophoresis is a fundamental tool of molecular biology that has been used extensively for the biochemical analysis of RNA-protein interactions. These interactions have been traditionally analyzed with polyacrylamide gels generated between two glass plates and samples electrophoresed vertically. However, polyacrylamide gels cast in trays and electrophoresed horizontally offers several advantages. For example, horizontal gels used to analyze complexes between fluorescent RNA substrates and specific proteins can be imaged multiple times as electrophoresis progresses. This provides the unique opportunity to monitor RNA-protein complexes at several points during the experiment. In addition, horizontal gel electrophoresis makes it possible to analyze many samples in parallel. This can greatly facilitate time course experiments as well as analyzing multiple reactions simultaneously to compare different components and conditions. Here we provide a detailed protocol for generating and using horizontal native gel electrophoresis for analyzing RNA-Protein interactions.

Native polyacrylamide gel electrophoresis is a fundamental tool for analyzing RNA-protein interactions. Traditionally most experiments have used vertical gels. However, horizontal gels provide several advantages, such as the opportunity to monitor complexes during electrophoresis. We provide a detailed protocol for generating and using horizontal native gel electrophoresis.

水平凝胶电泳用于增强蛋白质 -  RNA复合物的检测

Native polyacrylamide gel electrophoresis is a fundamental tool of molecular biology that has been used extensively for the biochemical analysis of ...

2 in 1: One-step Affinity Purification for the Parallel Analysis of Protein-Protein and Protein-Metabolite Complexes

Cellular processes are regulated by interactions between biological molecules such as proteins, metabolites, and nucleic acids. While the investigation of protein-protein interactions (PPI) is no novelty, experimental approaches aiming to characterize endogenous protein-metabolite interactions (PMI) constitute a rather recent development. Herein, we present a protocol that allows simultaneous characterization of the PPI and PMI of a protein of choice, referred to as bait. Our protocol was optimized for Arabidopsis cell cultures and combines affinity purification (AP) with mass spectrometry (MS)-based protein and metabolite detection. In short, transgenic Arabidopsis lines, expressing bait protein fused to an affinity tag, are first lysed to obtain a native cellular extract. Anti-tag antibodies are used to pull down protein and metabolite partners of the bait protein. The affinity-purified complexes are extracted using a one-step methyl tert-butyl ether (MTBE)/methanol/water method. Whilst metabolites separate into either the polar or the hydrophobic phase, proteins can be found in the pellet. Both metabolites and proteins are then analyzed by ultra-performance liquid chromatography-mass spectrometry (UPLC-MS or UPLC-MS/MS). Empty-vector (EV) control lines are used to exclude false positives. The major advantage of our protocol is that it enables identification of protein and metabolite partners of a target protein in parallel in near-physiological conditions (cellular lysate). The presented method is straightforward, fast, and can be easily adapted to biological systems other than plant cell cultures.

Protein-protein and protein-metabolite interactions are crucial for all cellular functions. Herein, we describe a protocol that allows parallel analysis of these interactions with a protein of choice. Our protocol was optimized for plant cell cultures and combines affinity purification with mass spectrometry-based protein and metabolite detection.

2在 1: 一步亲和纯化为蛋白质蛋白质和蛋白质代谢物的平行的分析

Cellular processes are regulated by interactions between biological molecules such as proteins, metabolites, and nucleic acids. While the investigation of ...

Use of Electron Paramagnetic Resonance in Biological Samples at Ambient Temperature and 77 K

The accurate and specific detection of reactive oxygen species (ROS) in different cellular and tissue compartments is essential to the study of redox-regulated signaling in biological settings. Electron paramagnetic resonance spectroscopy (EPR) is the only direct method to assess free radicals unambiguously. Its advantage is that it detects physiologic levels of specific species with a high specificity, but it does require specialized technology, careful sample preparation, and appropriate controls to ensure accurate interpretation of the data. Cyclic hydroxylamine spin probes react selectively with superoxide or other radicals to generate a nitroxide signal that can be quantified by EPR spectroscopy. Cell-permeable spin probes and spin probes designed to accumulate rapidly in the mitochondria allow for the determination of superoxide concentration in different cellular compartments.
In cultured cells, the use of cell permeable 1-hydroxy-3-methoxycarbonyl-2,2,5,5-tetramethylpyrrolidine (CMH) along with and without cell-impermeable superoxide dismutase (SOD) pretreatment, or use of cell-permeable PEG-SOD,&#160;allows for the differentiation of extracellular from cytosolic superoxide. The mitochondrial 1-hydroxy-4-[2-triphenylphosphonio)-acetamido]-2,2,6,6-tetramethyl-piperidine,1-hydroxy-2,2,6,6-tetramethyl-4-[2-(triphenylphosphonio)acetamido] piperidinium dichloride (mito-TEMPO-H) allows for measurement of mitochondrial ROS (predominantly superoxide).
Spin probes and EPR spectroscopy can also be applied to in vivo models. Superoxide can be detected in extracellular fluids such as blood and alveolar fluid, as well as tissues such as lung tissue. Several methods are presented to process and store tissue for EPR measurements and deliver intravenous 1-hydroxy-3-carboxy-2,2,5,5-tetramethylpyrrolidine (CPH) spin probe in vivo. While measurements can be performed at room temperature, samples obtained from in vitro and in vivo models can also be stored at -80 &#176;C and analyzed by EPR at 77 K. The samples can be stored in specialized tubing stable at -80 &#176;C and run at 77 K to enable a practical, efficient, and reproducible method that facilitates storing and transferring samples.

Electron paramagnetic resonance (EPR) spectroscopy is an unambiguous method to measure free radicals. The use of selective spin probes allows for detection of free radicals in different cellular compartments. We present a practical, efficient method to collect biological samples that facilitate treating, storing, and transferring samples for EPR measurements.

电子顺磁共振在生物样品环境温度和 77 k 的应用

The accurate and specific detection of reactive oxygen species (ROS) in different cellular and tissue compartments is essential to the study of ...

Single-step Purification of Macromolecular Complexes Using RNA Attached to Biotin and a Photo-cleavable Linker

For many years, the exceptionally strong and rapidly formed interaction between biotin and streptavidin has been successfully utilized for partial purification of biologically important RNA/protein complexes. However, this strategy suffers from one major disadvantage that limits its broader utilization: the biotin/streptavidin interaction can be broken only under denaturing conditions that also disrupt the integrity of the eluted complexes, hence precluding their subsequent functional analysis and/or further purification by other methods. In addition, the eluted samples are frequently contaminated with the background proteins that nonspecifically associate with streptavidin beads, complicating the analysis of the purified complexes by silver staining and mass spectrometry. To overcome these limitations, we developed a variant of the biotin/streptavidin strategy in which biotin is attached to an RNA substrate via a photo-cleavable linker and the complexes immobilized on streptavidin beads are selectively eluted to solution in a native form by long wave UV, leaving the background proteins on the beads. Shorter RNA binding substrates can be synthesized chemically with biotin and the photo-cleavable linker covalently attached to the 5&#39; end of the RNA, whereas longer RNA substrates can be provided with the two groups by a complementary oligonucleotide. These two variants of the UV-elution method were tested for purification of the U7 snRNP-dependent processing complexes that cleave histone pre-mRNAs at the 3&#39; end and they both proved to compare favorably to other previously developed purification methods. The UV-eluted samples contained readily detectable amounts of the U7 snRNP that was free of major protein contaminants and suitable for direct analysis by mass spectrometry and functional assays. The described method can be readily adapted for purification of other RNA binding complexes and used in conjunction with single- and double-stranded DNA binding sites to purify DNA-specific proteins and macromolecular complexes.

RNA/protein complexes purified using botin-streptavidin strategy are eluted to solution under denaturing conditions in a form unsuitable for further purification and functional analysis. Here, we describe a modification of this strategy that utilizes a photo-cleavable linker in RNA and a gentle UV-elution step, yielding native and fully functional RNA/protein complexes.

生物素和光裂解链接器所附 rna 对高分子配合物的单步纯化

For many years, the exceptionally strong and rapidly formed interaction between biotin and streptavidin has been successfully utilized for partial ...

Analysis of Protein Folding, Transport, and Degradation in Living Cells by Radioactive Pulse Chase

Radioactive pulse-chase labeling is a powerful tool for studying the conformational maturation, the transport to their functional cellular location, and the degradation of target proteins in live cells. By using short (pulse) radiolabeling times (&lt;30 min) and tightly controlled chase times, it is possible to label only a small fraction of the total protein pool and follow its folding. When combined with nonreducing/reducing SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoprecipitation with (conformation-specific) antibodies, folding processes can be examined in great detail. This system has been used to analyze the folding of proteins with a huge variation in properties such as soluble proteins, single and multi-pass transmembrane proteins, heavily N- and O-glycosylated proteins, and proteins with and without extensive disulfide bonding. Pulse-chase methods are the basis of kinetic studies into a range of additional features, including co- and posttranslational modifications, oligomerization, and polymerization, essentially allowing the analysis of a protein from birth to death. Pulse-chase studies on protein folding are complementary with other biochemical and biophysical methods for studying proteins in vitro by providing increased temporal resolution and physiological information. The methods as described within this paper are adapted easily to study the folding of almost any protein that can be expressed in mammalian or insect-cell systems.

Here we describe a protocol for a general pulse-chase method that allows the kinetic analysis of folding, transport, and degradation of proteins to be followed in live cells.

放射性脉冲追逐对活细胞蛋白质折叠、迁移和降解的分析

Radioactive pulse-chase labeling is a powerful tool for studying the conformational maturation, the transport to their functional cellular location, and ...