这项研究是由赠款全球前沿项目（2011-0031944）和KRIBB研究计划项目的智能合成生物学中心的支持。

1.生物传感器的制备<ol><li>按照先前建立的协议2 CFP-MBP-YFP-的His6 -构建质粒pET21a（+）。 </li><li>接种5毫升的Luria肉汤（LB）的与大肠杆菌 DE3菌株的单个菌落，并在37°C孵育16小时振荡。 </li><li>传输1毫升的O / N培养物在含有100ml LB和在振荡培养箱中于37℃孵育直到在600nm（OD 600）的光密度达到0.5（约3小时）的500ml烧瓶中。 </li><li>在1,000×g下收获在50ml的锥形管中的细胞通过离心在4℃下20分钟。 </li><li>用50毫升冰冷的蒸馏水（DW）和离心机快速重悬沉淀在每个管中，在1000×g下在4℃下20分钟。 </li><li>通过轻轻摇动直到溶液（电感受态细胞），用10％（V / V）甘油悬浮在50μl冰冷的DW粒料达到的OD 600 </suB&gt; 100。 </li><li>放置电感受态细胞的混合物（50微升细胞以OD的100 600）和质粒pET21a（+），10纳克- CFP-MBP-YFP-的His6在冰冷电穿孔管在电穿孔设备和电穿孔该混合物（18千伏/厘米，25μF）。 </li><li>迅速1毫升SOC培养基添加到反应杯和重悬轻轻细胞，然后回收在37℃下1小时，在15毫升的圆底管轻轻摇动。 </li><li>涂布在含有100微克/毫升氨苄青霉素的LB板中的细胞，并在37℃孵育12小时。 </li><li>使用循环隔离单菌落并接种菌落在10ml含有LB，在37℃100微克/ ml氨苄青霉素的摇动12小时的。 </li><li>将5ml种子培养添加到500毫升的LB含有100μg/ ml氨苄青霉素和孵育培养在37℃振荡培养箱中。 </li><li>添加0.5mM的异丙基β-D-硫代半乳糖苷（IPTG）的外径600到达时0.5孵育在37℃振荡培养箱中培养24小时。 </li><li>离心机的细胞在4500×g的20分钟（4℃），并轻轻取出上清。 </li><li>重新悬浮于5mL的结合缓冲液（20mM的Tris-HCl，pH值8.0，1mM的PMSF，0.5mM的EDTA，和1mM DTT）沉淀。 </li><li>超声处理上有六个10秒连发冰细胞在200-300瓦，下面的每个突发冷却10秒。 </li><li>离心裂解物在10,000× 克30分钟，在4℃以沉淀细胞碎片。转移上清液（可溶性蛋白）到一个新的收集管。 </li><li>为了实现FRET传感器蛋白质的亲和纯化，负载4毫升澄清的细胞裂解物上样到Ni-NTA亲和柱（5毫升体积），用快速蛋白液相层析（FPLC）18执行色谱测定。 </li><li>用洗涤缓冲液五个柱体积洗一次列I（50mM磷酸盐缓冲液，300毫氯化钠，10mM咪唑，pH 7.0）中。 </li><li>重复该洗涤步骤用洗涤缓冲液II五个柱体积（50mM磷酸盐缓冲液，300毫氯化钠，的20mM咪唑，pH 7.0）中。 </li><li>用洗脱缓冲液五个柱体积（50mM磷酸盐缓冲液，300毫氯化钠，500mM咪唑，pH 7.0）中洗脱传感器蛋白。 </li><li>浓缩和脱盐洗脱样品，填充高达20样品和离心机毫升浓缩器（10,000兆瓦膜尺寸），用于在3000×g下10分钟。重新填充用0.8％的磷酸盐缓冲盐水（PBS）浓缩。重复此步骤两次，第一次填充聚光用20ml样品，然后用PBS再填充。 </li><li>回收浓缩和脱盐传感器蛋白并将其储存在-80℃。 </li></ol> 2.使用FRET分析糖含量的测定注:FRET分析仪结构的细节在我们以前的工作7进行了描述。 <ol><li>制备含0.2 0.8％PBS的检测解决方案传感器蛋白质微米。 </li><li>打开FRET分析仪。按"向上"按钮2秒来校准最佳温度。用"UP"和"DOWN"按钮，将温度设置为53℃，并按下"SET"键。 </li><li>对于校准，请同时按住"UP"和"DOWN"按钮2秒。确认LED面板显示"CALIB"，然后按"SET"键。 </li><li>将只含PBS缓冲液进入分析仪的试管架，然后按"SET"键12.5×12.5×45毫米（长×宽×高）的长方体容器（试管）。 </li><li>以仅包含检测溶液（见2.1）不加糖（麦芽糖/蔗糖）更换试管，然后按"SET"键来校准基准。 </li><li>更换含10毫米糖检测解决方案，一个比色皿，并按下"SET"按钮。 </li><li>以确定一个饮料样品中的糖含量，把1毫升的饮料样品中的1.5毫升微量离心管中并离心16,000×g的1分钟。 注意:基于传感器FRET荧光测定具有不需要特殊的预处理样品的样品，因为只有1％（V / V）的优点包括在总体积。但是，我们建议去除可能影响荧光测定（ 例如，细胞，不溶性微粒，脂质，脂肪，或具有自发荧光的任何材料）的任何材料。此外，如果一强酸，强碱，清洁剂（洗涤剂），或乳化剂（乳化剂）的存在以高浓度并可能影响FRET生物传感器的特性，应当使用有机溶剂或除去中和剂。例如，当乳品脂肪和乳化剂选自冷冻点心消除，将样品以16,000×g离心以离心管30分钟，液体between中的底部沉积物和乳品脂肪顶层被提取。然后己烷等量加入，然后离心以15,000×g的30分钟，以消除脂质。 </li><li>用1-ml注射器除去上清液，并通过注射器过滤器（孔径为0.2μm）进行过滤。 </li><li>放置0.1毫升过滤饮料样品中含有0.9毫升PBS中，并涡旋轻轻1.5毫升离心管中。 注:这是正确稀释饮料样品的关键。在这种情况下，进行1000倍稀释，以使糖浓度将落在该装置的动态范围内。我们建议通过参照在饮料的标签的糖含量预先估计目标糖浓度。 </li><li>加入5微升稀释饮料样品（1％，V / V）的含有0.495毫升检测溶液的反应杯中。 </li><li>放置反应杯在FRET分析仪的反应杯保持器和预热样品溶液至53℃。 </li><li>按"SET"键来测量糖含量。 注意可以通过在五百三十五分之四百八十八毫微米7,8-读取比率评价使用配备有用于温度控制的珀尔帖装置的多标记平板读数器或荧光分光光度计FRET的测量。对于蔗糖的检测，请按照1.1的步骤2.12与CSY-LH传感器2。 </li></ol>

<ol>
	<li>Deuschle, K., Okumoto, S., Fehr, M., Looger, L. L., Kozhukh, L., Frommer, W. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Construction+and+optimization+of+a+family+of+genetically+encoded+metabolite+sensors+by+semirational+protein+engineering.">Construction and optimization of a family of genetically encoded metabolite sensors by semirational protein engineering.</a> Protein Sci. 14 (9), 2304-2314 (2005).</li><li>Ha, J. S., Song, J. J., Lee, Y. M., Kim, S. J., Sohn, J. H., Shin, C. S., Lee, S. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Design+and+application+of+highly+responsive+fluorescence+resonance+energy+transfer+biosensors+for+detection+of+sugar+in+living+Saccharomyces+cerevisiae+cells.">Design and application of highly responsive fluorescence resonance energy transfer biosensors for detection of sugar in living Saccharomyces cerevisiae cells.</a> Appl. Environ. Microbiol. 73 (22), 7408-7414 (2007).</li><li>Nagai, T., Yamada, S., Tominaga, T., Ichikawa, M., Miyawaki, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expanded+dynamic+range+of+fluorescent+indicators+for+Ca(2+)+by+circularly+permuted+yellow+fluorescent+proteins.">Expanded dynamic range of fluorescent indicators for Ca(2+) by circularly permuted yellow fluorescent proteins.</a> Proc. Natl. Acad. Sci. U.S.A. 101 (29), 10554-10559 (2004).</li><li>Okumoto, S., Looger, L. L., Micheva, K. D., Reimer, R. J., Smith, S. J., Frommer, W. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+glutamate+release+from+neurons+by+genetically+encoded+surface-displayed+FRET+nanosensors.">Detection of glutamate release from neurons by genetically encoded surface-displayed FRET nanosensors.</a> Proc. Natl. Acad. Sci. U.S.A. 102 (24), 8740-8745 (2005).</li><li>Merzlyakov, M., Li, E., Casas, R., Hristova, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spectral+F&#246;rster+resonance+energy+transfer+detection+of+protein+interactions+in+surface-supported+bilayers.">Spectral F&#246;rster resonance energy transfer detection of protein interactions in surface-supported bilayers.</a> Langmuir. 22 (16), 6986-6992 (2006).</li><li>Zhang, J., Campbell, R. E., Ting, A. Y., Tsien, R. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Creating+new+fluorescent+probes+for+cell+biology.">Creating new fluorescent probes for cell biology.</a> Nat. Rev. Mol. Cell Biol. 3 (12), 906-918 (2002).</li><li>Kim, H., Kim, H. S., Ha, J. S., Lee, S. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+portable+FRET+analyzer+for+rapid+detection+of+sugar+content.">A portable FRET analyzer for rapid detection of sugar content.</a> Analyst. 140 (10), 3384-3389 (2015).</li><li>Gam, J., Ha, J. -. S., Kim, H., Lee, D. -. H., Lee, J., Lee, S. -. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ratiometric+analyses+at+critical+temperatures+can+magnify+the+signal+intensity+of+FRET-based+sugar+sensors+with+periplasmic+binding+proteins.">Ratiometric analyses at critical temperatures can magnify the signal intensity of FRET-based sugar sensors with periplasmic binding proteins.</a> Biosens. Bioelectron. 72, 37-43 (2015).</li><li>Hessels, A. M., Merkx, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetically-encoded+FRET-based+sensors+for+monitoring+Zn2++in+living+cells.">Genetically-encoded FRET-based sensors for monitoring Zn2+ in living cells.</a> Metallomics. 7 (2), 258-266 (2015).</li><li>Song, Y., Yang, M., Wegner, S. V., Zhao, J., Zhu, R., Wu, Y., He, C., Chen, P. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+genetically+encoded+FRET+sensor+for+intracellular+heme.">A genetically encoded FRET sensor for intracellular heme.</a> ACS Chem. Biol. 10 (7), 1610-1615 (2015).</li><li>. Fluorescent Protein Guide: Biosensors Available from: <a target="_blank" href="https://www.addgene.org/fluorescent-proteins/biosensors/">https://www.addgene.org/fluorescent-proteins/biosensors/</a> (2015)</li><li>Rajendran, R., Rayman, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Point-of-care+blood+glucose+testing+for+diabetes+care+in+hospitalized+patients:+an+evidence-based+review.">Point-of-care blood glucose testing for diabetes care in hospitalized patients: an evidence-based review.</a> J. Diabetes Sci. Technol. 8 (6), 1081-1090 (2014).</li><li>Vyas, N. K., Vyas, M. N., Quiocho, F. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sugar+and+signal-transducer+binding+sites+of+the+Escherichia+coli+galactose+chemoreceptor+protein.">Sugar and signal-transducer binding sites of the Escherichia coli galactose chemoreceptor protein.</a> Science. 242, 1290-1295 (1988).</li><li>Leermakers, E. T. M., Felix, J. F., Erler, N. S., &#266;erimagi&#263;, A., Wijtzes, A. I., Hofman, A., Raat, H., Moll, H. A., Rivadeneira, F., Jaddoe, V. W., Franco, O. H., Kiefte-de Jong, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sugar-containing+beverage+intake+in+toddlers+and+body+composition+up+to+age+6+years:+The+Generation+R+Study.">Sugar-containing beverage intake in toddlers and body composition up to age 6 years: The Generation R Study.</a> Eur. J. Clin. Nutr. 69 (3), 314-321 (2015).</li><li>Shilts, M., Styne, D., Drake, C., Aden, C., Townsend, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fast+food,+fat+and+sugar+sweetened+beverage+items+are+related+to+children's+dietary+energy+density.">Fast food, fat and sugar sweetened beverage items are related to children's dietary energy density.</a> FASEB J. 29 (1), 731-736 (2015).</li><li>Larsson, S. C., &#197;kesson, A., Wolk, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sweetened+beverage+consumption+is+associated+with+increased+risk+of+stroke+in+women+and+men.">Sweetened beverage consumption is associated with increased risk of stroke in women and men.</a> J Nutr. 144 (6), 856-860 (2014).</li><li>Melkko, S., Neri, D., Vaillancourt, P. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calmodulin+as+an+affinity+purification+tag.">Calmodulin as an affinity purification tag.</a> E. coli Gene Expression Protocols. , 69-77 (2003).</li></ol>

作者什么都没有透露。

该协议允许饮料样品中的糖含量的快速和有效的量化，使用定做的FRET分析器7在用于FRET传感器的最佳温度。分析器被设计用最近开发的，廉价的405纳米波段紫外LED作为光源和两个光检测器与硅光电二极管。这个装置是更符合成本效益比其它可比的荧光计。该器件显示高检测灵敏度，在最佳的温度范围为FRET传感器测量两个发射波长带（530纳米和480纳米）的比率特别时。它的灵敏度和强度检测各种糖均优于荧光分光光度计装置7。 该协议的主要目标是支持与定制FRET分析仪基于FRET的传感器的广泛适用性。虽然分析仪间接测量通过FRET传感器糖度，我t是清楚的是，设备结合了一些FRET传感器，包括广泛延伸遗传工程配体特异性，模块化设计，传感器浓度无关的信号，和亚细胞的小分子的精确定位的好处。 FRET传感器实际用于检测多种小分子，包括离子9，血红素10，以及其他的。此外，超过20种FRET构造可以很容易找到，并通过非营利性的存款AddGene 11篇。 尽管FRET分析仪的适用性广，有与该装置的动作的两个主要问题。首先，由于该装置的操作比较简单，样品的预处理是影响检测的质量，除了在设备发生故障的情况下的关键步骤。在这个协议中，一个步骤（样品稀释）足以处理该显然透明液体样品和不含不溶杂色克莱斯。然而，其他的样品，可能需要额外的处理以除去不溶性材料，例如蜂窝式或脂质组分。可以影响的FRET信号的任何自身荧光颗粒应当也被除去，如所指出以下步骤2.7。其次，质量控制和连接与医院信息系统的接口需要与所有类型的床边血糖检测（POCT）工具12来加以解决。由于FRET分析仪的信号质量在很大程度上取决于FRET传感器的质量和在预处理步骤，需要定期质量控制检查，以确保测量保持定期质量控制数据分析的标准信号的范围内。该FRET传感器的稳定性和贮存期，这两者都是用于进一步可靠的应用必需的，应在质量控制检查进行调查。建立指导方针和发展适当的软件也可以解决的连接限制。当前版本FRET分析仪均配备了RS232连接远程命令行控制，但无线通信可能是分析仪的下一个版本，这将对医院信息系统改进的界面的功能。 然而，FRET传感器已经专为底物特异性，一种方法，通常允许更广泛的特异性2。因此，FRET信号可能遇到来自其它成分，包括其他类型的商业饮料糖的意外干扰。进一步的研究应该探索式传感器如何FRET各种糖混合物应对精确量化适量白糖。与生产饮料的公司合作将有助于确认含糖量，以校准FRET分析仪。 可以预期，与各种FRET传感器所提出的便携FRET设备将在POCT应用中使用。 POCT用于评估妊娠，血液血糖水平，生物标记物的蛋白质，传染性细菌，传染性病毒。 POCT方法具有快速的周转时间，并且通常表现出由于少数的处理步骤的低错误率。这些都是POCT在中心实验室检测方法的重要的优点。手持式便携POCT设备，如本文中所描述的装置中，已经吸引了越来越多，因为它们在食物的评估和血糖监测潜在的应用的关注。特别是，在糖尿病患者的血液样本的血糖监测需要一个快速，准确，和具有成本效益的POCT方法13。埃姆斯研究团队开发的第一血糖试纸后，在1965年（使用含有葡萄糖氧化酶条），几种技术都提出了血糖监测的目的12。 FRET的分析仪也可用于检测血液样品中的葡萄糖的血液和周质葡萄糖结合protei适当的预处理N（MglB）14基于FRET蛋白质。 需要对食品质量评估的简单，快速的方法。含糖饮料的消耗与多种疾病和综合症，如在儿童期15体重指数增加，小儿肥胖16和中风17的风险有关。理解这种连接就必须在饮料的糖组分的精确测量。因此，饮料的葡萄糖和果糖的浓度是感兴趣的涉及人体健康的科学家。这个协议说明了FRET分析仪具有最佳的温度控制的高度敏感的性能。该装置可与各种FRET传感器可用于检测各种小molecules-包括葡萄糖和果糖14,15。我们的便携式和可再充电装置，其具有10的电池寿命 - 20小时，这取决于加热协议，适用于POCT。其简单的操作方案MAKES设备易于使用，省去了复杂的工作人员培训。随着技术的改进，包括减少设备规模，预处理步骤最小化，并为现场使用实际要求鉴定，该设备将促进小规模的实验室环境基于FRET的研究开发。

荧光共振能量转移（FRET）已被广泛地用作生物统计传感器来检测小分子如糖，钙离子，氨基酸1-4。 FRET生物传感器含有荧光蛋白，青色荧光蛋白（国家重点计划）和黄色荧光蛋白（YFPs），其稠合到周质结合蛋白（的PBPs）的两端。糖类结合到位于FRET传感器的中间的PBPs，从而导致其随后改变在的PBPs的一端的两个荧光蛋白的距离和跃迁偶极取向传感器的结构变化。这种改变，通过测量EYFP（530纳米），ECFP（480纳米）的发光波长的比率使得能够糖含量的定量分析。由于高灵敏度，特异性，实时监测能力，和FRET生物传感器的响应时间快，这些传感器被广泛应用于环境，工业和医疗应用5。此外，比率式使用FRET生物传感器etric测量具有重要的实际的好处，因为它可以用来测量复杂生物样品，其中所述传感器的浓度不能被容易地控制和背景荧光总是存在于组件。 尽管基于FRET的传感器，用于定量可视化的这些优点，不完整的域名运动转移到荧光蛋白微小的结构性变化产生固有的弱信号强度。这个弱信号限制基于FRET的传感器，用于在体外或体内分析6的应用程序。因此，大多数FRET生物传感器需要使用昂贵的，高度敏感的设备。先前，我们开发了一种廉价和便携式FRET分析仪类似于现有荧光分析仪7的功能。在该装置中，廉价的405纳米波段紫外发光二极管（LED）作为光源，使第激发Ë荧光信号，更换昂贵的灯泡或激光。分析仪的检测系统的散热荧光信号高效率地聚焦到两个光电检测器与硅光电二极管。在最近的研究中，我们发现，在50检测温度的优化- 55°C可能显著放大比例FRET信号8。这个特定的温度信号增强，与定做的FRET分析仪一起使得能够快速且高灵敏度更一般诊断应用中的使用的FRET传感器。 在这个协议中，我们通过量化市售饮料的糖含量表现出最佳的FRET的温度条件下的FRET分析仪的通用性。这个协议提供了FRET设备操作的细节，以及传感器和样品制备的简要说明。我们预计，该报告将推动便携式的潜在应用分析器在小规模的实验室环境，并提供与基于FRET的生物传感器的廉价的现场诊断装置的进一步发展奠定了基础。

<table border="0" cellpadding="0" cellspacing="0" width="1297">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:227px;">LB</td>
			<td style="width:191px;">BD</td>
			<td style="width:119px;">#244620</td>
			<td style="width:760px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:227px;">isopropyl &#946;-D-thiogalactoside (IPTG)</td>
			<td style="width:191px;">Sigma</td>
			<td style="width:119px;">I6758</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:227px;">Ampicillin</td>
			<td style="width:191px;">Sigma</td>
			<td style="width:119px;">A9518</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:227px;">Tri-HCl</td>
			<td style="width:191px;">Bioneer</td>
			<td style="width:119px;">C-9006-1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:227px;">PMSF</td>
			<td style="width:191px;">Sigma</td>
			<td style="width:119px;">78830</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:227px;">EDTA</td>
			<td style="width:191px;">Bioneer</td>
			<td style="width:119px;">C-9007</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:227px;">DTT</td>
			<td style="width:191px;">Sigma</td>
			<td style="width:119px;">D0632</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:227px;">NaCl</td>
			<td style="width:191px;">Junsei</td>
			<td style="width:119px;">19015-0350</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:227px;">phosphate-buffered saline (PBS)</td>
			<td style="width:191px;">Gibco</td>
			<td style="width:119px;">70011-044</td>
			<td>0.8% NaCl, 0.02% KCl, 0.0144% Na2HPO4, 0.024% KH2OP4, pH 7.4</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:227px;">SOC</td>
			<td style="width:191px;"></td>
			<td style="width:119px;"></td>
			<td>2% tryptone, 0.5% Yeast extract, 10 mM NaCl, 2.5 mM KCl, 10 mM MGCl2, 20 mM Glucose</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:227px;">Resource Q</td>
			<td>Amersham Biosciences</td>
			<td style="width:119px;">17-1177-01</td>
			<td>6 &#215; 30 mm anion-exchange chromatography column&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:227px;">HisTrap HP1</td>
			<td>Amersham Biosciences</td>
			<td style="width:119px;">29-0510-21</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:227px;">Quartz cuvette</td>
			<td style="width:191px;">Sigma</td>
			<td style="width:119px;">Z802875</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:227px;">AK&#196;KTAFPLC</td>
			<td>Amersham Biosciences</td>
			<td style="width:119px;">18-1900-26</td>
			<td>a fast protein liquid chromatography (FPLC)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cary Eclipse</td>
			<td>VarianInc</td>
			<td style="width:119px;"></td>
			<td>a fluorescence spectrophotometer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:227px;">VICTOR&#160;&#160;</td>
			<td style="width:191px;">PerkinElmer</td>
			<td style="width:119px;">2030-0050</td>
			<td>a multilabel plate reader</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:227px;">E. coli JM109 (DE3)</td>
			<td style="width:191px;">Promega</td>
			<td style="width:119px;"></td>
			<td>Electrocompetent cells</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:227px;">A (Beverage)</td>
			<td style="width:191px;">Korea Yakult Co. (Korea)</td>
			<td style="width:119px;">Birak</td>
			<td>Fermented drinks</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:227px;">B (Beverage)</td>
			<td style="width:191px;">Lotte Foods (Korea)</td>
			<td style="width:119px;">Epro</td>
			<td>Soft drink</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:227px;">C (Beverage)</td>
			<td style="width:191px;">Lotte Foods (Korea)</td>
			<td style="width:119px;">Getoray</td>
			<td>Sports drink</td>
		</tr>
	</tbody>
</table>

highly sensitive and rapid fluorescence detection with a portable fret analyzer

在荧光共振能量转移最近的改进（FRET）传感器已经使它们的使用，以检测各种小分子，包括离子和氨基酸。然而，FRET传感器的先天弱信号强度，以防止在各个领域的应用并使得使用昂贵的，高端的荧光计必要的一个重大挑战。先前，我们构建可以特异性测量两个发射波长带（530和480纳米）的比率，以实现高的检测灵敏度具有成本效益的，高性能的FRET分析仪。最近，人们发现FRET传感器与细菌周质结合蛋白检测用在临界温度范围的50最高灵敏度的配体 - 55℃。这份报告描述了使用我们的便携式FRET分析仪具有特定的温度-FRET传感器市售饮料样品评估中含糖量的协议。我们的结果表明，附加的预热FRET的传感器的过程显著增加的FRET比率信号，以使糖含量更精确的测量。定做的FRET分析仪和传感器成功地应用到量化糖含量在三种类型的商业饮料。我们预计，设备的尺寸进一步降低和性能的提高将有利于环境中使用手持式分析仪，其中高端装备不可用。

以执行使用FRET分析糖含量的定量分析，必须建立一个拟合曲线推定从观察的FRET比率目标糖浓度。设r定义CFP的发光强度的比在480nm和在530nm（式1）中产生的YFP的发光强度。 <img alt="式（1）" src="/files/ftp_upload/54144/54144equation1.jpg" />可以通过观察FRET比r，在不同的糖浓度来生成（在53℃的CMY-BII）的FRET生物传感器的剂量-反应曲线。然后该曲线可以表示为S形S形曲线如下: <img alt="公式（2）" src="/files/ftp_upload/54144/54144equation2.jpg" />r max和R 最小值代表分别为0的糖浓度和饱和（1000μM）中，信号比; X 0表示，在50％的反应糖浓度; p表示的反应，这是接近1或-1的斜率。在本研究中，R 最大 ，R min时 ，X 0和p分别是4.256，2.672，71.779，和1。在模型拟合所用浓度范围为1微米至1000微米。 使用方程1和2，市售的饮料的糖含量是用FRET分析仪定量。两个麦芽糖FRET传感器进行了检查，以测试信号r，取决于各种温度2,8。第一FRET传感器，CMY-0，是由CFP，麦芽糖结合蛋白（MBP），和YFP的一个基本的基于FRET的传感器，其中nØ接头肽。第二传感器，CMY-BII，具有MBP和两个荧光蛋白2之间的丝氨酸-精氨酸接头。 如图1A示，CMY-0不在测定温度50℃以下观察到的，因为有0和1mM麦芽糖浓度之间没有信号差。两者的信号差异FRET传感器分别50和55℃之间最大（ 图1）8。为了量化三种市售饮料的糖含量，产生在53-℃下将CMY-BII传感器的剂量-响应曲线（ 图2A）和三个样品的麦芽糖含量是确定的转换的FRET比率进麦芽糖浓度。 为样品A是由谷物如水稻和大麦，这是重要的麦芽糖来源的，将样品预期含有相对高麦芽糖含量（平均11.892克/ 235毫升）（ <st荣&gt;图2A）。与此相反，样品C是一个运动饮料，有三种饮料中的最低麦芽糖（0.29ml克/ 250毫升）。这些结果表明，FRET的分析仪可以在最佳的温度下使用，以最大限度地提高的糖含量的定量，省去了昂贵的高端FRET的检测装置的需要。 <img alt="图1" src="/files/ftp_upload/54144/54144fig1.jpg" /> 图1.的FRET信号差介于0和1mM麦芽糖使用FRET分析仪在不同温度（A）的CMY-0传感器表明在温度低于50℃，在不同的麦芽糖浓度无信号差异。（B）中的CMY-BII传感器能区分在一个宽的温度范围在0和1毫麦芽糖之间的FRET信号差。 （ - 55℃50）在这两种情况下，该信号差异显着在特定的温度范围内增加。电子商务RROR条表示标准偏差。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54144/54144fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/54144/54144fig2.jpg" /> 图2.麦芽糖三市售的饮料。（A）剂量反应曲线量化的内容为CMY-BII（B）三种饮料样品麦芽糖含量进行定量。请注意，"总糖"表示所有的糖（包括麦芽糖）由饮料标签上的饮料制造商报告的数量。误差条表示标准偏差。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54144/54144fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about Highly Sensitive and Rapid Fluorescence Detection with a Portable FRET Analyzer at JoVE.com

Highly Sensitive and Rapid Fluorescence Detection with a Portable FRET Analyzer

这个协议描述了使用定制便携式FRET分析器荧光共振能量转移（FRET）的传感器数据的快速且高灵敏度定量。该装置被用于最大化的检测灵敏度，有利的糖含量的实际和有效的评估的临界温度范围内检测麦芽糖。

高度敏感和快速荧光检测用便携式分析仪FRET

Recent improvements in F&#246;rster resonance energy transfer (FRET) sensors have enabled their use to detect various small molecules including ions and ...

highly-sensitive-rapid-fluorescence-detection-with-portable-fret

Nanyang Technological University

Research

JoVE Journal

Biochemistry

9.0K 次观看.  Korea Research Institute of Bioscience and Biotechnology. 这个协议描述了使用定制便携式FRET分析器荧光共振能量转移（FRET）的传感器数据的快速且高灵敏度定量。该装置被用于最大化的检测灵敏度，有利的糖含量的实际和有效的评估的临界温度范围内检测麦芽糖。 

视频: Highly Sensitive and Rapid Fluorescence Detection with a Portable FRET Analyzer

High Precision FRET at Single-molecule Level for Biomolecule Structure Determination

A protocol on how to perform high-precision interdye distance measurements using F&#246;rster resonance energy transfer (FRET) at the single-molecule level in multiparameter fluorescence detection (MFD) mode is presented here. MFD maximizes the usage of all &#34;dimensions&#34; of fluorescence to reduce photophysical and experimental artifacts and allows for the measurement of interdye distance with an accuracy up to ~1 &#197; in rigid biomolecules. This method was used to identify three conformational states of the ligand-binding domain of the N-methyl-D-aspartate (NMDA) receptor to explain the activation of the receptor upon ligand binding. When comparing the known crystallographic structures with experimental measurements, they agreed within less than 3 &#197; for more dynamic biomolecules. Gathering a set of distance restraints that covers the entire dimensionality of the biomolecules would make it possible to provide a structural model of dynamic biomolecules.

A protocol for high-precision FRET experiments at the single molecule level is presented here. Additionally, this methodology can be used to identify three conformational states in the ligand-binding domain of the N-methyl-D-aspartate (NMDA) receptor. Determining precise distances is the first step towards building structural models based on FRET experiments.

单分子水平的高精度FRET用于生物分子结构测定

A protocol on how to perform high-precision interdye distance measurements using F&#246;rster resonance energy transfer (FRET) at the single-molecule ...

科研

生物化学

Regeneration of Arrayed Gold Microelectrodes Equipped for a Real-Time Cell Analyzer

The label-free cell-based assay is advantageous for biochemical study because of it does not require the use of experimental animals. Due to its ability to provide more dynamic information about cells under physiological conditions than classical biochemical assays, this label-free real-time cell assay based on the electric impedance principle is attracting more attention during the past decade. However, its practical utilization may be limited due to the relatively expensive cost of measurement, in which costly consumable disposable gold microchips are used for the cell analyzer. In this protocol, we have developed a general strategy to regenerate arrayed gold microelectrodes equipped for a commercial label-free cell analyzer. The regeneration process includes trypsin digestion, rinsing with ethanol and water, and a spinning step. The proposed method has been tested and shown to be effective for the regeneration and repeated usage of commercial electronic plates at least three times, which will help researchers save on the high running cost of real-time cell assays.

This protocol describes a general strategy to regenerate commercial arrayed gold microelectrodes equipped for a label-free cell analyzer aimed at saving on the high running costs ofmicrochip-based assays. The regeneration process includes trypsin digestion, rinsing with ethanol and water, and a spinning step, which enables repeated usage of microchips.

用于实时细胞分析仪的阵列金电极再生

The label-free cell-based assay is advantageous for biochemical study because of it does not require the use of experimental animals. Due to its ability ...

Rab10 Phosphorylation Detection by LRRK2 Activity Using SDS-PAGE with a Phosphate-binding Tag

Mutations in leucine-rich repeat kinase 2 (LRRK2) have been shown to be linked with familial Parkinson&#39;s disease (FPD). Since abnormal activation of the kinase activity of LRRK2 has been implicated in the pathogenesis of PD, it is essential to establish a method to evaluate the physiological levels of the kinase activity of LRRK2. Recent studies revealed that LRRK2 phosphorylates members of the Rab GTPase family, including Rab10, under physiological conditions. Although the phosphorylation of endogenous Rab10 by LRRK2 in cultured cells could be detected by mass spectrometry, it has been difficult to detect it by immunoblotting due to the poor sensitivity of currently available phosphorylation-specific antibodies for Rab10. Here, we describe a simple method of detecting the endogenous levels of Rab10 phosphorylation by LRRK2 based on immunoblotting utilizing sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) combined with a phosphate-binding tag (P-tag), which is N-(5-(2-aminoethylcarbamoyl)pyridin-2-ylmetyl)-N,N&#39;,N&#39;-tris(pyridin-2-yl-methyl)-1,3-diaminopropan-2-ol. The present protocol not only provides an example of the methodology utilizing the P-tag but also enables the assessment of how mutations as well as inhibitor treatment/administration or any other factors alter the downstream signaling of LRRK2 in cells and tissues.

The present study describes a simple method of detecting endogenous levels of Rab10 phosphorylation by leucine-rich repeat kinase 2.

用磷酸盐结合标记的 SDS-页 Rab10 磷酸化检测 LRRK2 活性

Mutations in leucine-rich repeat kinase 2 (LRRK2) have been shown to be linked with familial Parkinson&#39;s disease (FPD). Since abnormal activation of ...

Highly Sensitive and Quantitative Detection of Proteins and Their Isoforms by Capillary Isoelectric Focusing Method

Immunoblotting has become a routine technique in many laboratories for protein characterization from biological samples. The following protocol provides an alternative strategy, capillary isoelectric focusing (cIEF), with many advantages compared to conventional immunoblotting. This is an antibody-based, automated, rapid, and quantitative method in which a complete western blotting procedure takes place inside an ultrathin capillary. This technique does not require a gel to transfer to a membrane, stripping of blots, or x-ray films, which are typically required for conventional immunoblotting. Here, proteins are separated according to their charge (isoelectric point; pI), using less than a microliter (400 nL) of total protein lysate. After electrophoresis, proteins are immobilized onto the capillary walls by ultraviolet light treatment, followed by primary and secondary (horseradish peroxidase (HRP) conjugated) antibody incubation, whose binding is detected through enhanced chemiluminescence (ECL), generating a light signal that can be captured and recorded by a charge-coupled device (CCD) camera. The digital image can be analyzed and quantified (peak area) using software. This high throughput procedure can handle 96 samples at a time; is highly sensitive, with protein detection in the picogram range; and produces highly reproducible results because of automation. All of these aspects are extremely valuable when the quantity of samples (e.g., tissue samples and biopsies) is a limiting factor. The technique has wider applications as well, including screening of drugs or antibodies, biomarker discovery, and diagnostic purposes.

Capillary isoelectric focusing is an antibody-based, ultrasensitive, high throughput technique, enabling detailed characterization of proteins and their isoforms from extremely small biological samples. The following describes a protocol for detection and quantification of specific proteins and their isoforms in an automated and robotized manner.

毛细管电聚焦法对蛋白质及其亚型的高灵敏度和定量检测

Immunoblotting has become a routine technique in many laboratories for protein characterization from biological samples. The following protocol provides ...

Detection of Detergent-sensitive Interactions Between Membrane Proteins

Our ability to explore protein-protein interactions is the key to understanding regulatory connections in the cell. However, detection of protein-protein interactions in many cases is associated with significant experimental challenges. In particular, sorting receptors interact with their protein cargo in the lumen of the membrane compartments often in a detergent-sensitive fashion, making co-immunoprecipitation of these proteins unusable. Binding of the sorting receptor sortilin to glucose transporter GLUT4 may serve as an example of weak luminal interactions between membrane proteins. Here, we describe a fast, simple, and inexpensive assay to validate the interaction between sortilin and GLUT4. For that, we have designed and chemically synthesized the myc-tagged peptide corresponding to the potential sortilin-binding epitope in the luminal part of GLUT4. Sortilin tagged with six histidines was expressed in mammalian cells, and isolated from cell lysates using Cobalt beads. Sortilin immobilized on the beads was incubated with the peptide solution at different pH values, and the eluted material was analyzed by Western blotting. This assay can be easily adapted to study other detergent-sensitive protein-protein interactions.

We describe a protocol for detection of detergent-sensitive interactions between membrane proteins using binding of the sorting receptor, sortilin, to the first luminal loop of the glucose transporter protein, GLUT4, as an example.

膜蛋白对洗涤剂敏感相互作用的检测

Our ability to explore protein-protein interactions is the key to understanding regulatory connections in the cell. However, detection of protein-protein ...

Detection of Small GTPase Prenylation and GTP Binding Using Membrane Fractionation and  GTPase-linked Immunosorbent Assay

The Rho GTPase family belongs to the Ras superfamily and includes approximately 20 members in humans. Rho GTPases are important in the regulation of diverse cellular functions, including cytoskeletal dynamics, cell motility, cell polarity, axonal guidance, vesicular trafficking, and cell cycle control. Changes in Rho GTPase signaling play an essential regulatory role in many pathological conditions, such as&#160;cancer, central nervous system diseases, and immune system-dependent diseases. The posttranslational modification of Rho GTPases (i.e., prenylation by mevalonate pathway intermediates) and GTP binding are key factors which affect the activation of this protein. In this paper, two essential and simple methods are provided to detect a broad range of Rho GTPase prenylation and GTP binding activities. Details of the technical procedures that have been used are explained step by step in this manuscript.

Here we describe a protocol to investigate the prenylation and guanosine-5'-triphosphate (GTP)-loading of Rho GTPase. This protocol consists of two detailed methods, namely membrane fractionation and a GTPase-linked immunosorbent assay. The protocol can be used for measuring the prenylation and GTP loading of different other small GTPases.

膜分馏和 gtpase-关联免疫吸附法检测小 gtpase 预基化和 gtp 结合

The Rho GTPase family belongs to the Ras superfamily and includes approximately 20 members in humans. Rho GTPases are important in the regulation of ...

Rapid Fluorescence-based Characterization of Single Extracellular Vesicles in Human Blood with Nanoparticle-tracking Analysis

Extracellular vesicles (EVs), including exosomes, are specialized membranous nano-sized vesicles found in bodily fluids that are constitutively released from many cell types and play a pivotal role in regulating cell-cell communication and a diverse range of biological processes. Many different methods for the characterization of EVs have been described. However, most of these methods have the disadvantage that the preparation and characterization of the samples are very time-consuming, or it is extremely difficult to analyze specific markers of interest due to their small size and due to the lack of discrete populations. While methods for analysis of EVs have been considerably improved over the last decade, there is still no standardized method for characterization of single EVs. Here, we demonstrate a semi-automated method for characterization of single EVs by fluorescence-based nanoparticle-tracking analysis. The protocol that is presented addresses the common problem of many researchers in this field and provides the complete workflow for rapid isolation of EVs and characterization with PKH67, a general cell membrane linker, as well as with specific surface markers such as CD63, CD9, vimentin, and lysosomal-associated membrane protein 1 (LAMP-1). The presented results show a high level of reproducibility, as confirmed by other methods, such as Western blotting. In the conducted experiments, we exclusively used EVs isolated from human serum samples, but this method is also suitable for plasma or other body fluids and can be adjusted for characterization of EVs from cell culture supernatants. Irrespective of the future progress of research on EV biology, the protocol that is presented here provides a rapid and reliable method for rapid characterization of single EVs with specific markers.

In this protocol, we describe the complete workflow for rapid isolation of extracellular vesicles from human whole blood and characterization of specific markers by fluorescence-based nanoparticle-tracking analysis. The presented results show a high level of reproducibility and can be adjusted to cell culture supernatants.

基于荧光的人体血液单细胞外囊快速表征

Extracellular vesicles (EVs), including exosomes, are specialized membranous nano-sized vesicles found in bodily fluids that are constitutively released ...

Detection of Protein Ubiquitination Sites by Peptide Enrichment and Mass Spectrometry

The posttranslational modification of proteins by the small protein ubiquitin is involved in many cellular events. After tryptic digestion of ubiquitinated proteins, peptides with a diglycine remnant conjugated to the epsilon amino group of lysine (&#39;K-&#949;-diglycine&#39; or simply &#39;diGly&#39;) can be used to track back the original modification site. Efficient immunopurification of diGly peptides combined with sensitive detection by mass spectrometry has resulted in a huge increase in the number of ubiquitination sites identified up to date. We have made several improvements to this workflow, including offline high pH reverse-phase fractionation of peptides prior to the enrichment procedure, and the inclusion of more advanced peptide fragmentation settings in the ion routing multipole. Also, more efficient cleanup of the sample using a filter-based plug in order to retain the antibody beads results in a greater specificity for diGly peptides. These improvements result in the routine detection of more than 23,000 diGly peptides from human cervical cancer cells (HeLa) cell lysates upon proteasome inhibition in the cell. We show the efficacy of this strategy for in-depth analysis of the ubiquitinome profiles of several different cell types and of in vivo samples, such as brain tissue. This study presents an original addition to the toolbox for protein ubiquitination analysis to uncover the deep cellular ubiquitinome.

We present a method for the purification, detection, and identification of diGly peptides that originate from ubiquitinated proteins from complex biological samples. The presented method is reproducible, robust, and outperforms published methods with respect to the level of depth of the ubiquitinome analysis.

通过肽浓缩和质谱检测蛋白质泛化位点检测

The posttranslational modification of proteins by the small protein ubiquitin is involved in many cellular events. After tryptic digestion of ...

Study on the Metabolism of Six Systemic Insecticides in a Newly Established Cell Suspension Culture Derived from Tea (Camellia Sinensis L.) Leaves

A platform for studying insecticide metabolism using in vitro tissues of tea plant was developed. Leaves from sterile tea plantlets were induced to form loose callus on Murashige and Skoog (MS) basal media with the plant hormones 2,4-dichlorophenoxyacetic acid (2,4-D, 1.0 mg L-1) and kinetin (KT, 0.1 mg L-1). Callus formed after 3 or 4 rounds of subculturing, each lasting 28 days. Loose callus (about 3 g) was then inoculated into B5 liquid media containing the same plant hormones and was cultured in a shaking incubator (120 rpm) in the dark at 25 &#177; 1 &#176;C. After 3&#8722;4 subcultures, a cell suspension derived from tea leaf was established at a subculture ratio ranging between 1:1 and 1:2 (suspension mother liquid: fresh medium). Using this platform, six insecticides (5 &#181;g mL-1 each thiamethoxam, imidacloprid, acetamiprid, imidaclothiz, dimethoate, and omethoate) were added into the tea leaf-derived cell suspension culture. The metabolism of the insecticides was tracked using liquid chromatography and gas chromatography. To validate the usefulness of the tea cell suspension culture, the metabolites of thiamethoxan and dimethoate present in treated cell cultures and intact plants were compared using mass spectrometry. In treated tea cell cultures, seven metabolites of thiamethoxan and two metabolites of dimethoate were found, while in treated intact plants, only two metabolites of thiamethoxam and one of dimethoate were found. The use of a cell suspension simplified the metabolic analysis compared to the use of intact tea plants, especially for a difficult matrix such as tea.

Study on the Metabolism of Six Systemic Insecticides in a Newly Established Cell Suspension Culture Derived from Tea Camellia Sinensis L. Leaves

This work presents a protocol for establishing a cell suspension culture derived from tea (Camellia sinensis L.) leaves that can be used to study the metabolism of external compounds that can be taken up by the whole plant, such as insecticides.

从茶中提取的新建细胞悬浮培养基中六种系统杀虫剂的代谢研究(山茶西宁西斯L.叶

A platform for studying insecticide metabolism using in vitro tissues of tea plant was developed. Leaves from sterile tea plantlets were induced to form ...

Extremely Rapid and Specific Metabolic Labelling of RNA In Vivo with 4-Thiouracil (Ers4tU)

The nucleotide analogue, 4-thiouracil (4tU), is readily taken up by cells and incorporated into RNA as it is transcribed in vivo, allowing isolation of the RNA produced during a brief period of labelling. This is done by attaching a biotin moiety to the incorporated thio group and affinity purifying, using streptavidin coated beads. Achieving a good yield of pure, newly synthesized RNA that is free of pre-existing RNA makes shorter labelling times possible and permits increased temporal resolution in kinetic studies. This is a protocol for very specific, high yield purification of newly synthesized RNA. The protocol presented here describes how RNA is extracted from the yeast Saccharomyces cerevisiae. However, the protocol for purification of thiolated RNA from total RNA should be effective using RNA from any organism once it has been extracted from the cells. The purified RNA is suitable for analysis by many widely used techniques, such as reverse transcriptase-qPCR, RNA-seq and SLAM-seq. The specificity, sensitivity and flexibility of this technique allow unparalleled insights into RNA metabolism.

Extremely Rapid and Specific Metabolic Labelling of RNA In Vivo with 4-Thiouracil Ers4tU

The use of thiolated uracil to sensitively and specifically purify newly transcribed RNA from the yeast Saccharomyces cerevisiae.

极快速和特殊的代谢标签的RNA在Vivo与4-Thiouracil （Ers4tU）

The nucleotide analogue, 4-thiouracil (4tU), is readily taken up by cells and incorporated into RNA as it is transcribed in vivo, allowing isolation of ...