这项工作是由马普学会的支持。

质粒1。创作<ol><li>亚克隆的cDNA为每个感兴趣的蛋白到两个pLuc和pYFP载体，用标准的分子生物学技术（ 图1）。有关详细方案见绿等[13]。 </li><li>序列中的所有构造，以验证所感兴趣的蛋白质在帧与吕克/ YFP序列中间没有终止密码子。 </li><li>如期望的，以确认该融合蛋白保留生物活性进行功能测定。在这里呈现的情况下YFP融合蛋白的亚细胞定位是确定通过荧光显微镜。 </li><li>通过设计相关的目标信号进入pLuc和pYFP表达载体使表达质粒适当吕克和YFP控制蛋白质。在这里介绍的情况下，通过插入一个核定位信号转换成pLuc和pYFP向量生成核针对性吕克和YFP结构。 </li><li>使其中卢克和YFP融合成单个多肽阳性对照构建体。在这里呈现的情况下，产生的阳性对照，其中，YFP编码序列被插入到pLuc矢量。 </li><li>选择一个中性填料质粒被用于均衡的DNA的质量中的转染混合物。使用的质粒不具有真核启动子，因此，将无转录活性的哺乳动物细胞，如细菌克隆载体中。 </li></ol> 2制备的DNA混合料<ol><li>估计所有在第1基于260nm处的吸光度所描述的质粒的浓度（1吸光度单位相当于DNA的50微克/毫升）。通过由650沓乘以碱基对的数量确定每种质粒的分子量。使用浓度和分子量来计算每个质粒制备的摩尔浓度。稀释质粒DNA制备到36 Nm的浓度。这些都将是工作股将用于制备用于转染的DNA的混合物。 </li><li>制备含20微升的水至终​​体积1800纳克填料质粒的对照DNA的组合。 </li><li>制备含有5微升的pLuc控制结构的控制DNA混合物。添加填料质粒带来的总DNA质量1800纳克。加水使最终体积为20微升。 </li><li>制备含有5微升pLuc控制结构和5μlpYFP控制结构的控制DNA混合物。添加填料质粒带来的总DNA质量1800纳克。加水使最终体积为20微升。 </li><li>制备含有5微升阳性对照结构的控制DNA混合物。添加填料质粒带来的总DNA质量1800纳克。加水使最终体积为20微升。 </li><li>准备以下的DNA混合，以测试对于感兴趣，X.蛋白质的同源二聚化对于每一个DNA结合结构5微升的相关pLuc构造和5μl的PY的FP构造。添加填料质粒带来的总DNA质量1800纳克。加水使最终体积为20微升。 A）pLuc控制和pYFP-X B）pLuc-X和pYFP控制 C）pLuc-X和pYFP-X </li><li>准备以下的DNA混合来测试一对感兴趣的蛋白质之间的相互作用，X和Y。对于每一个DNA结合结构5微升的相关pLuc构建和5微升的pYFP的构建。添加填料质粒带来的总DNA质量1800纳克。加水使最终体积为20微升。 A）pLuc控制和pYFP-X B）pLuc-X和pYFP控制 C）pLuc控制和pYFP-Y D）pLuc-Y和pYFP控制 E）pLuc-X和pYFP-Y F）pLuc-Y和pYFP-X </li></ol> 3，转染<ol><li>收获汇合从一个75 厘米烧瓶HEK293细胞。稀释的细胞总数的10％为13 ml培养介质中。免除130微升细胞悬浮液到一个白色的各孔中清澈见底的96孔培养板。培养细胞24小时。 </li><li>计算由乘以3的DNA混合物的数量被转染井的数量。带来的无血清培养基中至室温。制备含6.3μL无血清培养基中，每孔0.18微升转染试剂预混液。涡旋混合并在室温下孵育5分钟。 </li><li>通过加入2微升的DNA混合，以20微升无血清培养基/转染试剂主混合物的制备转染混合物。不要旋涡。在室温下孵育10分钟。 </li><li>转染三口井，每个转染混合物分装6.5微升转染混合物，每孔。培养的细胞用于进一步的36-48小时。 </li></ol> BRET信号4。测量<ol><li>溶解活细胞荧光素酶的底物在34毫克/毫升DMSO中通过涡旋。 </li><li>在基材的稀释介质P稀重组活细胞荧光素酶底物在1:1,000重新加热到37℃。让50微升底物稀释介质，每孔。旋涡混匀。沉淀物可能形成，但不会影响检测干扰。 </li><li>从96孔板中吸出培养基。免除加入50μl稀释的活细胞荧光素酶底物在每孔中。培养细胞至少2小时（最多24个小时）。 </li><li>从96孔板中除去盖子，孵育板10分钟，在室温下的发光计内。 </li><li>从吕克和YFP一个度量发射阱的时间。使用过滤器阻挡波长大于470纳米测量从吕克排放。使用500-600纳米的带通滤波器测量从YFP的发射。集成发射信号在10秒内。 </li></ol> 5。数据分析<ol><li>从3井只收填料质粒的平均吕克排放量读数。从所有其他吕克排放读数减去这个值来产生背景消减吕克排放值。</li><li>从3井只收填料质粒平均YFP的发射读数。从所有其他YFP的发射读数减去这个值来产生扣除背景的YFP的排放值。 </li><li>对于每孔，通过扣除背景的吕克排放值除以扣除背景的YFP的排放值，让裸BRET比例。 </li><li>平均从转染仅pLuc-对照质粒三个孔中的未校正的BRET比率。减去这个值从所有其他未校正的BRET比率，得到校正后的BRET比率。 </li><li>对于每个校正BRET比例的剩余组，平均从3转染的孔中的值，以获得最终的BRET比率。 </li></ol>

<ol>
	<li>Suter, B., Kittanakom, S., Stagljar, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two-hybrid+technologies+in+proteomics+research.">Two-hybrid technologies in proteomics research.</a> Curr Opin Biotechnol. 19, 316-323 (2008).</li><li>Gingras, A. C., Gstaiger, M., Raught, B., Aebersold, R. <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+protein+complexes+using+mass+spectrometry.">Analysis of protein complexes using mass spectrometry.</a> Nat Rev Mol Cell Biol. 8, 645-654 (2007).</li><li>Berggard, T., Linse, S., James, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+for+the+detection+and+analysis+of+protein-protein+interactions.">Methods for the detection and analysis of protein-protein interactions.</a> Proteomics. 7, 2833-2842 (2007).</li><li>Ciruela, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescence-based+methods+in+the+study+of+protein-protein+interactions+in+living+cells.">Fluorescence-based methods in the study of protein-protein interactions in living cells.</a> Curr Opin Biotechnol. 19, 338-343 (2008).</li><li>Pfleger, K. D., Eidne, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Illuminating+insights+into+protein-protein+interactions+using+bioluminescence+resonance+energy+transfer.">Illuminating insights into protein-protein interactions using bioluminescence resonance energy transfer.</a> BRET). Nat Methods. 3, 165-174 (2006).</li><li>Boute, N., Jockers, R., Issad, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+use+of+resonance+energy+transfer+in+high-throughput+screening.">The use of resonance energy transfer in high-throughput screening.</a> BRET versus FRET. Trends Pharmacol Sci. 23, 351-354 (2002).</li><li>Pfleger, K. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extended+bioluminescence+resonance+energy+transfer+(eBRET)+for+monitoring+prolonged+protein-protein+interactions+in+live+cells.">Extended bioluminescence resonance energy transfer (eBRET) for monitoring prolonged protein-protein interactions in live cells.</a> Cell Signal. 18, 1664-1670 (2006).</li><li>Kocan, M., Dalrymple, M., Seeber, R., Feldman, B., Pfleger, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhanced+BRET+technology+for+the+monitoring+of+agonist-induced+and+agonist-independent+interactions+between+GPCRs+and+&#946;-arrestins.">Enhanced BRET technology for the monitoring of agonist-induced and agonist-independent interactions between GPCRs and &#946;-arrestins.</a> Frontiers in Endocrinology. 1, (2011).</li><li>Boute, N., Pernet, K., Issad, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monitoring+the+activation+state+of+the+insulin+receptor+using+bioluminescence+resonance+energy+transfer.">Monitoring the activation state of the insulin receptor using bioluminescence resonance energy transfer.</a> Mol Pharmacol. 60, 640-645 (2001).</li><li>Perroy, J., Pontier, S., Charest, P. G., Aubry, M., Bouvier, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Real-time+monitoring+of+ubiquitination+in+living+cells+by+BRET.">Real-time monitoring of ubiquitination in living cells by BRET.</a> Nat Methods. 1, 203-208 (2004).</li><li>Roduit, R., Escher, P., Schorderet, D. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mutations+in+the+DNA-binding+domain+of+NR2E3+affect+in+vivo+dimerization+and+interaction+with+CRX.">Mutations in the DNA-binding domain of NR2E3 affect in vivo dimerization and interaction with CRX.</a> PLoS One. 4, (2009).</li><li>Deriziotis, P., Fisher, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurogenomics+of+speech+and+language+disorders:+the+road+ahead.">Neurogenomics of speech and language disorders: the road ahead.</a> Genome Biol. 14, 204 (2013).</li><li>Green, M. R., Sambrook, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Molecular Cloning: A Laboratory Manual. , (2012).</li><li>Lai, C. S., Fisher, S. E., Hurst, J. A., Vargha-Khadem, F., Monaco, A. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+forkhead-domain+gene+is+mutated+in+a+severe+speech+and+language+disorder.">A forkhead-domain gene is mutated in a severe speech and language disorder.</a> Nature. 413, 519-523 (2001).</li><li>Fisher, S. E., Scharff, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FOXP2+as+a+molecular+window+into+speech+and+language.">FOXP2 as a molecular window into speech and language.</a> Trends Genet. 25, 166-177 (2009).</li><li>Graham, S. A., Fisher, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Decoding+the+genetics+of+speech+and+language.">Decoding the genetics of speech and language.</a> Curr Opin Neurobiol. 23, 43-51 (2013).</li><li>O'Roak, B. 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=Exome+sequencing+in+sporadic+autism+spectrum+disorders+identifies+severe+de+novo+mutations.">Exome sequencing in sporadic autism spectrum disorders identifies severe de novo mutations.</a> Nat Genet. 43, 585-589 (2011).</li><li>Horn, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+FOXP1+deletions+in+three+unrelated+patients+with+mental+retardation+and+significant+speech+and+language+deficits.">Identification of FOXP1 deletions in three unrelated patients with mental retardation and significant speech and language deficits.</a> Hum Mutat. 31, 1851-1860 (2010).</li><li>Hamdan, F. 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=De+novo+mutations+in+FOXP1+in+cases+with+intellectual+disability,+autism,+and+language+impairment.">De novo mutations in FOXP1 in cases with intellectual disability, autism, and language impairment.</a> Am J Hum Genet. 87, 671-678 (2010).</li><li>Talkowski, M. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sequencing+chromosomal+abnormalities+reveals+neurodevelopmental+loci+that+confer+risk+across+diagnostic+boundaries.">Sequencing chromosomal abnormalities reveals neurodevelopmental loci that confer risk across diagnostic boundaries.</a> Cell. 149, 525-537 (2012).</li><li>Li, S., Weidenfeld, J., Morrisey, E. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcriptional+and+DNA+binding+activity+of+the+Foxp1/2/4+family+is+modulated+by+heterotypic+and+homotypic+protein+interactions.">Transcriptional and DNA binding activity of the Foxp1/2/4 family is modulated by heterotypic and homotypic protein interactions.</a> Mol Cell Biol. 24, 809-822 (2004).</li><li>MacDermot, K. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+FOXP2+truncation+as+a+novel+cause+of+developmental+speech+and+language+deficits.">Identification of FOXP2 truncation as a novel cause of developmental speech and language deficits.</a> Am J Hum Genet. 76, 1074-1080 (2005).</li><li>Vernes, S. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+genetic+analysis+of+mutations+implicated+in+a+human+speech+and+language+disorder.">Functional genetic analysis of mutations implicated in a human speech and language disorder.</a> Hum Mol Genet. 15, 3154-3167 (2006).</li></ol>

作者宣称，他们有没有竞争的财务权益。

融合蛋白表达结构的设计是建立在BRET分析的关键步骤。在这里介绍的实验中，感兴趣的蛋白被融合至萤光素酶或YFP的C-末端。它也是可能的，并且可能是必要的，融合蛋白的荧光素酶/ YFP的N-末端。对于某些蛋白，融合仅可在无论是N-或C-末端，以避免蛋白质结构和功能的破坏所接受。此外，对于跨膜蛋白，其中N和C末端位于不同亚细胞，荧光素酶/ YFP蛋白必须融合到这是在同一货舱的互动合作伙伴?...

既古典联动和下一代人类疾病的测序分析都透出参与了一系列生物途径的蛋白的临床意义。这是通常的情况是，之前，他们在这些研究中鉴定，一直存在的这些蛋白质的生物学作用很小或没有调查。一个卓有成效的途径，开始探索的兴趣蛋白质的生物学功能是识别哪些其他蛋白质它与它的生理范围内进行交互。表征分子网络以这种方式提供了深入了解人类表型相关的生物途径。 最常用的大规模筛选方法确定候选互动合作伙伴的利益蛋白酵母双杂交筛选1和质谱为基础的蛋白质组学2。这些方法可以是非常成功的提示潜在的相互作用的蛋白质，但ARË容易受到假阳性结果。因此，确认确定了酵母双杂交或质谱筛查的交互的需要的交互验证利用第二技术。典型的免疫共沉淀或下拉测定法用于这一目的3。使用这样的技术进行验证的一个缺点是对于细胞裂解，从而破坏了细胞内的条件，可能是维持一定的蛋白相互作用所必需的要求。第二个缺点是软弱或短暂蛋白相互作用可在洗涤步骤被打乱。此外，这些测定要求显著动手的时间，在可同时处理的样本的数目是有限的，并且通常需要试剂和方案的费时的优化。 克服某些与免疫共沉淀实验相关联的问题，有几个实验已经基于荧光和biolum开发inescent蛋白质，可以在活细胞中使用。第一个这样的测定法是基于荧光（或福斯特）共振能量转移（FRET），能量的两种荧光蛋白之间的非辐射转移具有重叠发射光谱和激发光谱4。能量转移的效率很大程度上距离相关，因此观察的FRET现象，要求供体和受体荧光基团是近在咫尺。为了测试两种蛋白质的利益之间的相互作用，一种蛋白质被表达为融合与供体荧光团（通常青色荧光蛋白; CFP）和第二作为融合与受体荧光团（通常黄色荧光蛋白; YFP）。这两种蛋白的利益之间的相互作用可能带来的供体和受体荧光基团足够接近的能量转移发生，这将导致光的发射从YFP的受体相对的CFP供体可衡量的增加。 FRET已经成功地检测蛋白质-蛋白质相互作用中的活细胞4。利用FRET用于检测蛋白质 - 蛋白质相互作用的主要缺点是需要外部照明的供体荧光团的激发。外部照明效果在高背景的发射信号，受不必要的激励和两个供体和受体荧光基团的光漂白。这些效应降低了测定，用于检测蛋白质 - 蛋白质相互作用的灵敏度。 从外部照明克服了高背景问题FRET化验的修改是生物发光共振能量转移（BRET）检测5,6。在BRET系统中的供体荧光团所取代的荧光素酶的酶。因此，能量的受体荧光团的激发在系统内生成的荧光素酶底物的氧化，使外部照明是不必要的。在最该测定的通用结构中，供体是海肾reniformis荧光素酶和受体是YFP（用于替代供体和受体蛋白的讨论见PFLEGER 等人5）。因此，在这种系统中，感兴趣的蛋白融合至萤光素酶和潜在的相互作用蛋白对YFP，或反之亦然。在BRET实验需要加入腔肠素作为底物为荧光素酶。由于腔肠素是细胞渗透性的，因此能够在活细胞中进行BRET分析。然而，本机腔肠素是在水溶液中不稳定，和腔肠素的酶无关的击穿既降低衬底可用于测定中的浓度，并产生自发光，从而降低了荧光素酶活性测量的灵敏度。在活细胞使用BRET已经促进了保护coelenterazines，这是在水溶液中稳定，但受细胞内酯酶裂解的发展穿过细胞膜扩散，以产生电池7内部有源腔肠素后第下列添加底物对细胞表达荧光素酶和YFP融合蛋白，由蛋白质 - 蛋白质相互作用产生的能量转移是通过从荧光素酶和YFP监测排放量化。因为蛋白质的相互作用可直接在活细胞中的多孔板进行监测时，BRET分析构成一个简单的，可扩展的方法，用于检测推定的相互作用是成本和时间效益。 除了验证蛋白质组学筛选研究确定假定的干扰作用，在BRET系统也可以用来从感兴趣的蛋白质前生化和结构的研究而产生的测试候选干扰作用。一旦一个蛋白质 - 蛋白质相互作用的存在已经建立（或者通过使用BRET分析或通过其它技术），存在对于BRET分析为EMP是潜在loyed进一步表征的相互作用。例如，相互作用的区域可通过产生蛋白质的截短形式的映射值，并在特定的相互作用残基的参与可以通过创建点突变来证明。此外，在蛋白质-蛋白质相互作用的翻译后修饰，或小分子（如药物或配体）的调节作用可以调查8-10。 BRET的检测也有一个调查患者的DNA鉴定突变的巨大潜力。在情况下，一个突变的致病作用已经确立，学习使用BRET可以揭示更多关于11表型的分子病因学蛋白-蛋白相互作用的基因突变的影响。由于新一代测序方法的出现，人们越来越普遍的几个潜在损害性突变个体内被发现的，在这种情况下，目前还不清楚这是勒VANT的表型12。在这种情况下，BRET分析可能是在评价突变对蛋白功能的影响有价值的，因此它们对疾病的相关性。

<table border="0" cellpadding="0" cellspacing="0" width="1333">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:276px;">Nanodrop 8000</td>
			<td style="width:123px;">Nanodrop</td>
			<td style="width:155px;"></td>
			<td style="width:780px;">Any spectrophotometer capable of reading absorbances at 260 nm will be suitable. Determining the molecular mass of the plasmid is crucial for calculating DNA quantities to be used in transfection mixes.</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;">96 microwell plates, flat bottom, white</td>
			<td>Greiner Bio One</td>
			<td>655098</td>
			<td style="width:780px;">White plates reduce the crosstalk between wells and maximize the sensitivity of luminescence detection. Clear-bottomed wells allow monitoring of cell density. Plates must be suitable for cell culture. If using a top-reading luminometer the plate lid should be taken off.</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:276px;">Infinite F200Pro plate reader with control software</td>
			<td>TECAN</td>
			<td></td>
			<td style="width:780px;">Use the &#39;Blue 1&#39; and &#39;Green 1&#39; filters for luminescence measurement and the filter sets and dichoic mirror for GFP for fluorescence measurement. Any top-reading plate reader with capability of measuring dual-color luminescence and fluorescence is suitable.&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">pLuc, pYFP, positive control plasmid</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>Plasmids available from the authors upon request.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">pGEM-3Zf(+)</td>
			<td>Promega</td>
			<td>P2271</td>
			<td>Filler plasmid for equilization of DNA mass in transfection mixes. Any plasmid lacking a eukaryotic promoter would be suitable.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">HEK293 cells</td>
			<td>ECACC</td>
			<td>85120602</td>
			<td>Other cell lines that transfect with reasonable efficiency may be suitable.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">DMEM, high glucose, with phenol red</td>
			<td>Gibco</td>
			<td>41966</td>
			<td style="width:780px;">This is the medium used for culturing HEK293 cells. Warm in 37 &#176;C waterbath before use. If using a different cell line, replace the growth medium described here with cell-line specific medium.</td>
		</tr>
		<tr height="67">
			<td height="67" style="height:67px;">DMEM, high glucose, no phenol red (substrate dilution medium)</td>
			<td>Gibco</td>
			<td>21063</td>
			<td style="width:780px;">This is the substrate dilution medium used for dilution of the luciferase substrate (EnduRen) as it does not contain phenol red, which reduces the sensitivity of the assay. Contains HEPES to maintain correct pH during luminescence measurements while cells are out of the CO2 incubator. Warm in 37 &#176;C waterbath before use.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">OptiMEM</td>
			<td>Gibco</td>
			<td>31985</td>
			<td style="width:780px;">OptiMEM is used for dilution of GeneJuice transfection reagent. Other serum-free media would also be suitable. Warm to room temperature before use.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fetal bovine serum</td>
			<td>Gibco</td>
			<td>10270</td>
			<td>For supplementation of cell culture media at a concentration of 10% v/v.</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;">GeneJuice transfection reagent</td>
			<td>Novagen</td>
			<td>70967</td>
			<td style="width:780px;">If using a cell line other than HEK293, it may be necessary to adjust the ratio of Genejuice transfection reagent to DNA in the transfection mixes. Other transfection reagents may be used. If using an alternative transfection reagent, it may be necessary to optimize the amount of DNA used in the transfection mixes based on manufacturer&#39;s instructions.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DMSO</td>
			<td>Sigma</td>
			<td>D2650</td>
			<td style="width:780px;">Use sterile DMSO that is suitable for tissue culture.&#160;</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;">EnduRen live-cell substrate</td>
			<td>Promega</td>
			<td>E6481</td>
			<td style="width:780px;">Reconstitute EnduRen at 34 mg/ml in DMSO. Upon dilution of EnduRen in culture medium a precipitate may form. This will not interfere with the assay. Store reconstituted EnduRen at -20 &#176;C, and avoid multiple freeze-thaw cycles. Ensure that reconstituted EnduRen is completely thawed before diluting it in culture medium.</td>
		</tr>
	</tbody>
</table>

investigating protein-protein interactions in live cells using bioluminescence resonance energy transfer

基于生物发光共振能量转移（BRET）测定提供一种灵敏和可靠的手段来监测蛋白质 - 蛋白质相互作用中的活细胞。 BRET是能量从一个"供体"萤光素酶的非辐射转移到一个"受体"荧光蛋白。在此测定法中最常用的结构中，供体是海肾reniformis荧光素酶和受体是黄色荧光蛋白（YFP）。因为能量转移的效率很大程度上距离相关，观察的BRET现象，要求供体和受体在靠近。为了测试两种蛋白质在培养的哺乳动物细胞的兴趣之间的相互作用，一种蛋白质被表达为融合与荧光素酶和第二作为融合与YFP。这两种蛋白的利益之间的相互作用可能使供体和受体充分接近能量转移发生。与其他技术相比在调查的蛋白 - 蛋白间相互作用的BRET分析是敏感的，只需要很少的操作时间和一些试剂，并且能够检测这是短暂而微弱的，或者依赖于活细胞内发现的生化环境的相互作用。因此，它是用于确认通过酵母双杂交或质谱蛋白质组学研究提示推定的相互作用的理想方法，此外它是非常适合用于映射相互作用区域，评估的翻译后修饰的蛋白 - 蛋白相互作用的影响，并评估在患者的DNA鉴定的突变的影响。

在BRET实验的原理图示于图2。整个这里介绍的实验中所用的测定法，设置在图3中描绘。从转染的细胞的荧光素酶-YFP融合蛋白的强的BRET信号的检测证实了能量转移是可观察在本实验装置（ 图4）。 我们的研究主要集中在FOXP家庭在大脑发育的转录抑制的作用。影响FOXP2导致言语和语言障碍的一种罕见的，其中最突出的特点是发展语言?...

Watch this Scientific Journal Video about Investigating Protein-protein Interactions in Live Cells Using Bioluminescence Resonance Energy Transfer at JoVE.com

Investigating Protein-protein Interactions in Live Cells Using Bioluminescence Resonance Energy Transfer

蛋白质之间的相互作用是十分重要的所有细胞过程。使用生物发光共振能量转移，一对蛋白质之间的相互作用可以在活细胞中，并实时监测。此外，潜在的致病突变的效果进行评估。

研究蛋白质 - 蛋白质相互作用活细胞使用生物发光共振能量转移

Assays based on Bioluminescence Resonance Energy Transfer (BRET) provide a sensitive and reliable means to monitor protein-protein interactions in live ...

investigating-protein-protein-interactions-live-cells-using

Research

JoVE Journal

Biology

22.8K 次观看.  Max Planck Institute for Psycholinguistics. 蛋白质之间的相互作用是十分重要的所有细胞过程。使用生物发光共振能量转移，一对蛋白质之间的相互作用可以在活细胞中，并实时监测。此外，潜在的致病突变的效果进行评估。 

视频: Investigating Protein-protein Interactions in Live Cells Using Bioluminescence Resonance Energy Transfer

Microfluidic Co-culture of Epithelial Cells and Bacteria for Investigating Soluble Signal-mediated Interactions

The human gastrointestinal (GI) tract is a unique environment in which intestinal epithelial cells and non-pathogenic (commensal) bacteria coexist. It has been proposed that the microenvironment that the pathogen encounters in the commensal layer is important in determining the extent of colonization. Current culture methods for investigating pathogen colonization are not well suited for investigating this hypothesis as they do not enable co-culture of bacteria and epithelial cells in a manner that mimics the GI tract microenvironment. Here we describe a microfluidic co-culture model that enables independent culture of eukaryotic cells and bacteria, and testing the effect of the commensal microenvironment on pathogen colonization. The co-culture model is demonstrated by developing a commensal Escherichia coli biofilm among HeLa cells, followed by introduction of enterohemorrhagic E. coli (EHEC) into the commensal island, in a sequence that mimics the sequence of events in GI tract infection.

This protocol describes a microfluidic co-culture model for simultaneous and localized culture of epithelial cells and bacteria. This model can be used for investigating the role of different soluble molecular signals on pathogenesis as well as screen the effectiveness of putative probiotic bacterial strains.

微流体调查可溶性信号介导的相互作用的上皮细胞和细菌的共同文化

The human gastrointestinal (GI) tract is a unique environment in which intestinal epithelial cells and non-pathogenic (commensal) bacteria coexist. It has ...

科研

生物学

Investigating Tissue- and Organ-specific Phytochrome Responses using FACS-assisted Cell-type Specific Expression Profiling in Arabidopsis thaliana

Light mediates an array of developmental and adaptive processes throughout the life cycle of a plant. Plants utilize light-absorbing molecules called photoreceptors to sense and adapt to light. The red/far-red light-absorbing phytochrome photoreceptors have been studied extensively. Phytochromes exist as a family of proteins with distinct and overlapping functions in all higher plant systems in which they have been studied1. Phytochrome-mediated light responses, which range from seed germination through flowering and senescence, are often localized to specific plant tissues or organs2. Despite the discovery and elucidation of individual and redundant phytochrome functions through mutational analyses, conclusive reports on distinct sites of photoperception and the molecular mechanisms of localized pools of phytochromes that mediate spatial-specific phytochrome responses are limited. We designed experiments based on the hypotheses that specific sites of phytochrome photoperception regulate tissue- and organ-specific aspects of photomorphogenesis, and that localized phytochrome pools engage distinct subsets of downstream target genes in cell-to-cell signaling. We developed a biochemical approach to selectively reduce functional phytochromes in an organ- or tissue-specific manner within transgenic plants. Our studies are based on a bipartite enhancer-trap approach that results in transactivation of the expression of a gene under control of the Upstream Activation Sequence (UAS) element by the transcriptional activator GAL43. The biliverdin reductase (BVR) gene under the control of the UAS is silently maintained in the absence of GAL4 transactivation in the UAS-BVR parent4. Genetic crosses between a UAS-BVR transgenic line and a GAL4-GFP enhancer trap line result in specific expression of the BVR gene in cells marked by GFP expression4. BVR accumulation in Arabidopsis plants results in phytochrome chromophore deficiency in planta5-7. Thus, transgenic plants that we have produced exhibit GAL4-dependent activation of the BVR gene, resulting in the biochemical inactivation of phytochrome, as well as GAL4-dependent GFP expression. Photobiological and molecular genetic analyses of BVR transgenic lines are yielding insight into tissue- and organ-specific phytochrome-mediated responses that have been associated with corresponding sites of photoperception4, 7, 8. Fluorescence Activated Cell Sorting (FACS) of GFP-positive, enhancer-trap-induced BVR-expressing plant protoplasts coupled with cell-type-specific gene expression profiling through microarray analysis is being used to identify putative downstream target genes involved in mediating spatial-specific phytochrome responses. This research is expanding our understanding of sites of light perception, the mechanisms through which various tissues or organs cooperate in light-regulated plant growth and development, and advancing the molecular dissection of complex phytochrome-mediated cell-to-cell signaling cascades.

Investigating Tissue- and Organ-specific Phytochrome Responses using FACS-assisted Cell-type Specific Expression Profiling in Arabidopsis thaliana

The molecular basis of spatial-specific phytochrome responses is being investigated using transgenic plants that exhibit tissue- and organ-specific phytochrome deficiencies. The isolation of specific cells exhibiting induced phytochrome chromophore depletion by Fluorescence-Activated Cell Sorting followed by microarray analyses is being utilized to identify genes involved in spatial-specific phytochrome responses.

调查组织和器官特定的光敏色素反应，使用流式细胞仪辅助细胞类型特定的表达谱拟南芥</em

Light mediates an array of developmental and adaptive processes throughout the life cycle of a plant. Plants utilize light-absorbing molecules called ...

In-vivo Detection of Protein-protein Interactions on Micro-patterned Surfaces

Unraveling the interaction network of molecules in-vivo is key to understanding the mechanisms that regulate cell function and metabolism. A multitude of methodological options for addressing molecular interactions in cells have been developed, but most of these methods suffer from being rather indirect and therefore hardly quantitative. On the contrary, a few high-end quantitative approaches were introduced, which however are difficult to extend to high throughput. To combine high throughput capabilities with the possibility to extract quantitative information, we recently developed a new concept for identifying protein-protein interactions (Schwarzenbacher et al., 2008). Here, we describe a detailed protocol for the design and the construction of this system which allows for analyzing interactions between a fluorophore-labeled protein ("prey") and a membrane protein ("bait") in-vivo. Cells are plated on micropatterned surfaces functionalized with antibodies against the bait exoplasmic domain. Bait-prey interactions are assayed via the redistribution of the fluorescent prey. The method is characterized by high sensitivity down to the level of single molecules, the capability to detect weak interactions, and high throughput capability, making it applicable as screening tool.

In-vivo Detection of Protein-protein Interactions on Micro-patterned Surfaces

This video shows experiments with subsequent analysis of protein-protein interactions by the use of micro-patterned surfaces. The approach offers the possibility to detect protein interactions in living cells and combines high throughput capabilities with the possibility to extract quantitative information.

微图案的表面上的蛋白质-蛋白质相互作用的体内检测

Unraveling the interaction network of molecules in-vivo is key to understanding the mechanisms that regulate cell function and metabolism. A multitude of ...

Imaging Protein-protein Interactions in vivo

Protein-protein interactions are a hallmark of all essential cellular processes. However, many of these interactions are transient, or energetically weak, preventing their identification and analysis through traditional biochemical methods such as co-immunoprecipitation. In this regard, the genetically encodable fluorescent proteins (GFP, RFP, etc.) and their associated overlapping fluorescence spectrum have revolutionized our ability to monitor weak interactions in vivo using F&ouml;rster resonance energy transfer (FRET)1-3. Here, we detail our use of a FRET-based proximity assay for monitoring receptor-receptor interactions on the endothelial cell surface.

Imaging Protein-protein Interactions in vivo

This protocol describes how to image protein-protein interactions using a FRET-based proximity assay.

成像蛋白质 - 蛋白质相互作用在体内</em

Protein-protein interactions are a hallmark of all essential cellular processes. However, many of these interactions are transient, or energetically weak, ...

Real-time Monitoring of Ligand-receptor Interactions with Fluorescence Resonance Energy Transfer

FRET is a process whereby energy is non-radiatively transferred from an excited donor molecule to a ground-state acceptor molecule through long-range dipole-dipole interactions1. In the present sensing assay, we utilize an interesting property of PDA: blue-shift in the UV-Vis electronic absorption spectrum of PDA (Figure 1) after an analyte interacts with receptors attached to PDA2,3,4,7. This shift in the PDA absorption spectrum provides changes in the spectral overlap (J) between PDA (acceptor) and rhodamine (donor) that leads to changes in the FRET efficiency. Thus, the interactions between analyte (ligand) and receptors are detected through FRET between donor fluorophores and PDA. In particular, we show the sensing of a model protein molecule streptavidin. We also demonstrate the covalent-binding of bovine serum albumin (BSA) to the liposome surface with FRET mechanism. These interactions between the bilayer liposomes and protein molecules can be sensed in real-time. The proposed method is a general method for sensing small chemical and large biochemical molecules. Since fluorescence is intrinsically more sensitive than colorimetry, the detection limit of the assay can be in sub-nanomolar range or lower8. Further, PDA can act as a universal acceptor in FRET, which means that multiple sensors can be developed with PDA (acceptor) functionalized with donors and different receptors attached on the surface of PDA liposomes.

We demonstrate FRET between conjugated polymer polydiacetylene (PDA) and fluorophore attached to the surface of PDA liposomes for the sensing of biomolecules. PDA liposomes also contained receptor molecules on their surfaces for biomolecules to be used as probes. Ligand-receptor interactions lead to changes in the FRET efficiency between the fluorophore and PDA which is the basis of the sensing mechanism.

实时监控的配体 - 受体相互作用的荧光共振能量转移

FRET is a process  whereby energy is non-radiatively transferred from an excited donor molecule to  a ground-state acceptor molecule through long-range ...

Protein-protein Interactions Visualized by Bimolecular Fluorescence Complementation in Tobacco Protoplasts and Leaves

Many proteins interact transiently with other proteins or are integrated into multi-protein complexes to perform their biological function. Bimolecular fluorescence complementation (BiFC) is an in vivo method to monitor such interactions in plant cells. In the presented protocol the investigated candidate proteins are fused to complementary halves of fluorescent proteins and the respective constructs are introduced into plant cells via agrobacterium-mediated transformation. Subsequently, the proteins are transiently expressed in tobacco leaves and the restored fluorescent signals can be detected with a confocal laser scanning microscope in the intact cells. This allows not only visualization of the interaction itself, but also the subcellular localization of the protein complexes can be determined. For this purpose, marker genes containing a fluorescent tag can be coexpressed along with the BiFC constructs, thus visualizing cellular structures such as the endoplasmic reticulum, mitochondria, the Golgi apparatus or the plasma membrane. The fluorescent signal can be monitored either directly in epidermal leaf cells or in single protoplasts, which can be easily isolated from the transformed tobacco leaves. BiFC is ideally suited to study protein-protein interactions in their natural surroundings within the living cell. However, it has to be considered that the expression has to be driven by strong promoters and that the interaction partners are modified due to fusion of the relatively large fluorescence tags, which might interfere with the interaction mechanism. Nevertheless, BiFC is an excellent complementary approach to other commonly applied methods investigating protein-protein interactions, such as coimmunoprecipitation, in vitro pull-down assays or yeast-two-hybrid experiments.

Formation of protein complexes in vivo can be visualized by bimolecular fluorescence complementation. Interaction partners are fused to complementary parts of fluorescent tags and transiently expressed in tobacco leaves, resulting in a reconstituted fluorescent signal upon close proximity of the two proteins.

蛋白质 - 蛋白质相互作用的双分子荧光互补的烟草原生质体和叶片可视化

Many proteins interact transiently with other proteins or are integrated into multi-protein complexes to perform their biological function. Bimolecular ...

Probing High-density Functional Protein Microarrays to Detect Protein-protein Interactions

High-density functional protein microarrays containing ~4,200 recombinant yeast proteins are examined for kinase protein-protein interactions using an affinity purified yeast kinase fusion protein containing a V5-epitope tag for read-out. Purified kinase is obtained through culture of a yeast strain optimized for high copy protein production harboring a plasmid containing a Kinase-V5 fusion construct under a GAL inducible promoter. The yeast is grown in restrictive media with a neutral carbon source for 6 hr followed by induction with 2% galactose. Next, the culture is harvested and kinase is purified using standard affinity chromatographic techniques to obtain a highly purified protein kinase for use in the assay. The purified kinase is diluted with kinase buffer to an appropriate range for the assay and the protein microarrays are blocked prior to hybridization with the protein microarray. After the hybridization, the arrays are probed with monoclonal V5 antibody to identify proteins bound by the kinase-V5 protein. Finally, the arrays are scanned using a standard microarray scanner, and data is extracted for downstream informatics analysis1,2 to determine a high confidence set of protein interactions for downstream validation in vivo.

Using protein microarrays containing nearly the entire S. cerevisiae proteome is probed for rapid unbiased interrogation of thousands of protein-protein interactions in parallel. This method can be utilized for protein-small molecule, posttranslational modification, and other assays in high-throughput.

探讨高密度功能蛋白微阵列来检测蛋白质 - 蛋白质相互作用

High-density functional protein microarrays containing ~4,200 recombinant yeast proteins are examined for kinase protein-protein interactions using an ...

Real Time Measurements of Membrane Protein:Receptor Interactions Using Surface Plasmon Resonance (SPR)

Protein-protein interactions are pivotal to most, if not all, physiological processes, and understanding the nature of such interactions is a central step in biological research. Surface Plasmon Resonance (SPR) is a sensitive detection technique for label-free study of bio-molecular interactions in real time. In a typical SPR experiment, one component (usually a protein, termed 'ligand') is immobilized onto a sensor chip surface, while the other (the 'analyte') is free in solution and is injected over the surface. Association and dissociation of the analyte from the ligand are measured and plotted in real time on a graph called a sensogram, from which pre-equilibrium and equilibrium data is derived. Being label-free, consuming low amounts of material, and providing pre-equilibrium kinetic data, often makes SPR the method of choice when studying dynamics of protein interactions. However, one has to keep in mind that due to the method's high sensitivity, the data obtained needs to be carefully analyzed, and supported by other biochemical methods. 
SPR is particularly suitable for studying membrane proteins since it consumes small amounts of purified material, and is compatible with lipids and detergents. This protocol describes an SPR experiment characterizing the kinetic properties of the interaction between a membrane protein (an ABC transporter) and a soluble protein (the transporter's cognate substrate binding protein).

Real Time Measurements of Membrane Protein:Receptor Interactions Using Surface Plasmon Resonance SPR

Surface Plasmon Resonance (SPR) is a label-free method for detecting bio-molecular interactions in real time. Herein, a protocol for a membrane protein:receptor interaction experiment is described, while discussing the pros and cons of the technique.

膜蛋白的实时测量:受体相互作用使用表面等离子体共振（SPR）

Protein-protein interactions are pivotal to most, if not all, physiological processes, and  understanding the nature of such interactions is a central ...

Identifying Protein-protein Interaction Sites Using Peptide Arrays

Protein-protein interactions mediate most of the processes in the living cell and control homeostasis of the organism. Impaired protein interactions may result in disease, making protein interactions important drug targets. It is thus highly important to understand these interactions at the molecular level. Protein interactions are studied using a variety of techniques ranging from cellular and biochemical assays to quantitative biophysical assays, and these may be performed either with full-length proteins, with protein domains or with peptides. Peptides serve as excellent tools to study protein interactions since peptides can be easily synthesized and allow the focusing on specific interaction sites. Peptide arrays enable the identification of the interaction sites between two proteins as well as screening for peptides that bind the target protein for therapeutic purposes. They also allow high throughput SAR studies. For identification of binding sites, a typical peptide array usually contains partly overlapping 10-20 residues peptides derived from the full sequences of one or more partner proteins of the desired target protein. Screening the array for binding the target protein reveals the binding peptides, corresponding to the binding sites in the partner proteins, in an easy and fast method using only small amount of protein.
In this article we describe a protocol for screening peptide arrays for mapping the interaction sites between a target protein and its partners. The peptide array is designed based on the sequences of the partner proteins taking into account their secondary structures. The arrays used in this protocol were Celluspots arrays prepared by INTAVIS Bioanalytical Instruments. The array is blocked to prevent unspecific binding and then incubated with the studied protein. Detection using an antibody reveals the binding peptides corresponding to the specific interaction sites between the proteins.

Peptide array screening is a high throughput assay for identifying protein-protein interaction sites. This allows mapping multiple interactions of a target protein and can serve as a method for identifying sites for inhibitors that target a protein. Here we describe a protocol for screening and analyzing peptide arrays.

鉴定蛋白质相互作用的网站使用多肽阵列

Protein-protein interactions mediate most of the processes in the living cell and control homeostasis of the organism. Impaired protein interactions may ...

A Graphical User Interface for Software-assisted Tracking of Protein Concentration in Dynamic Cellular Protrusions

Filopodia are dynamic, finger-like cellular protrusions associated with migration and cell-cell communication. In order to better understand the complex signaling mechanisms underlying filopodial initiation, elongation and subsequent stabilization or retraction, it is crucial to determine the spatio-temporal protein activity in these dynamic structures. To analyze protein function in filopodia, we recently developed a semi-automated tracking algorithm that adapts to filopodial shape-changes, thus allowing parallel analysis of protrusion dynamics and relative protein concentration along the whole filopodial length. Here, we present a detailed step-by-step protocol for optimized cell handling, image acquisition and software analysis. We further provide instructions for the use of optional features during image analysis and data representation, as well as troubleshooting guidelines for all critical steps along the way. Finally, we also include a comparison of the described image analysis software with other programs available for filopodia quantification. Together, the presented protocol provides a framework for accurate analysis of protein dynamics in filopodial protrusions using image analysis software.

We present a software solution for semi-automated tracking of relative protein concentration along the length of dynamic cellular protrusions.

软件辅助跟踪动态细胞突变中蛋白质浓度的图形用户界面

Filopodia are dynamic, finger-like cellular protrusions associated with migration and cell-cell communication. In order to better understand the complex ...

研究蛋白质 - 蛋白质相互作用活细胞使用生物发光共振能量转移

摘要

探索更多视频

此视频中的章节