Name: investigating protein-protein interactions in live cells using bioluminescence resonance energy transfer
Uploaded: 2014-05-26T13:45:00
Description: Watch this Scientific Journal Video about Investigating Protein-protein Interactions in Live Cells Using Bioluminescence Resonance Energy Transfer at JoVE.com

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。 </...

<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>

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 ...

Research

JoVE Journal

Biology

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

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

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

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

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

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

Imaging Protein-protein Interactions in vivo

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

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

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

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)

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

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

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

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 ...