这项工作是从美国国立卫生补助（1R21NS073097-01）和1R01DK082884（JSD）为国家机构授予PIER11VCTR的和基金会的资金支持。

1。生成稳定的多克隆细胞系<ol><li>高品质的稳定表达细胞系感兴趣的KIR通道的建立，是一个发展一个强大的高通量筛选分析的重要的第一步。构K +通道的过度表达可导致细胞死亡的途径，稳定的细胞系的检测性能的变性和损失的激活。为了避免这些潜在的问题，并提供了一种方便的内部控制，研发分析（见下文），四环素诱导表达系统建议8。 </li><li>文化的父母T-REX-HEK293细胞用标准技术在B-培养基（DMEM生长培养基中含有10％FBS，50 U / ml的青霉素，50微克/毫升链霉素和5微克/毫升杀稻瘟菌素小号）。使用早期传代细胞（ 如 3-4个通道，因为从液氮存储解冻），转染和稳定的克隆选择。 </li><li>铠甲4万T-REX-HEK293细胞中75cm 2的烧瓶中，使烧瓶中的约80％汇合翌日。培养过夜，在5％CO 2培养箱中，在37℃下</li><li>根据生产商的方案，使用10-15微克pcDNA5/TO-Kir DNA与Lipofectamine LTX / Plus试剂转染细胞。 5小时后，更换转染培养基与B介质。 </li><li>在转染24小时后，与乙介质含有250微克/毫升潮霉素（BH-培养基），以开始稳定的克隆选择替换B介质。饲料的细胞每2-3天用新鲜的BH介质。 </li><li>大于90％的细胞死亡在未来7天里，留下一小部分的稳定转染细胞。允许殖民地成长为拆分前的10-14天，至175厘米2瓶扩展。 </li><li>冻存，冻结3×10 6细胞/ ml在含有45％的培养基，空调BH-培养基，45％不含抗生素，含有血清的DMEM和10％DMSO。冻结过夜的细胞在-80℃下的细胞冷冻容器中，然后移动到液氮用于长期存储。解冻细胞从液氮中，使用Invitrogen公司推荐的协议。需要注意的是细胞的生存能力将是较低的，当它们存在的烧瓶中，大于75％汇合在加工时从冻结。 </li></ol> 2。生成稳定的单克隆细胞株<ol><li>洗了一个铺满75厘米2烧瓶稳定的多克隆细胞有价无HBSS。加入1毫升的胰蛋白酶和孵育3-5分钟，在5％CO 2培养箱中，在37℃下向烧瓶中加入5毫升的BH介质抑制胰蛋白酶活性和磨碎反复（ 例如 ，5次），以完全解离的细胞。细胞在显微镜下仔细检查，以确保悬挂几乎完全由单细胞组成。这获得单克隆细胞系的可能性将增加。 </li><li>确定细胞密度和稀释细胞每20μl的浓度为0.7的悬浮液。使用多通道移液管，移液管将20μl细胞悬浮液到BD PureCoat胺涂覆（或等效的聚-D-赖氨酸涂层）384孔板的每个孔中。原则上，70％的井应该接受一个细胞，这些电镀条件。因此，在384 - 孔板应包含超过200克隆分析。继续培养细胞在37℃下，在5％CO 2培养箱中</li><li>一个星期后，检查板在显微镜下的井含单菌落。请注意自己的立场盖永久性标记，以备将来参考。也一定要注意排除包含多个殖民地的井。这是最容易识别的外观以及从多个侧面的生长。要知道，蒸发趋于发生，更迅速地从井附近的板的边缘。添加BH介质蒸发发生了显着的井。否则，它是不必要的吨Ø饲料的细胞。 </li><li>在未来7至10天，更频繁的监测井。细胞准备分裂时，井至少50％汇合。 </li><li>分裂细胞，重复的384孔板;继续在培养的单克隆线与Tl +流和其他用于测定的一个板。吸液从孔中含有感兴趣的细胞系的介质。二价无HBSS洗涤细胞，加入20μl的胰蛋白酶到各孔和板转移到37℃的培养箱中15-20分钟。移动板回到细胞培养罩后，各孔中加入20μl的BH-介质，，和磨碎几次以解离细胞。检查井在显微镜下，以确保细胞是完全解离的。这可能需要多轮的移液操作，会紧紧地贴壁的细胞。一旦细胞解离，10μl的每个细胞悬浮液转移到一个新的BD PureCoat胺涂层38的井重复4 - 孔板。一定要注意之间的关系源和复制目的地井，表现出强大的基尔通道活动（见下文）的克隆，可以追溯到原来的井。到原始的源以及喂剩余的细胞并继续在培养在5％CO 2培养箱中，在37℃下加入20μl的BH介质</li><li>允许在目标板的细胞粘附和生长长达一个星期。一旦大部分克隆达到至少50％汇合时，他们可以评估为吉珥通道的活性，使用的Tl +通量测定如下所述。 </li><li>检测的前一天，吸出培养基，取而代之的是与BH介质中含有10％透析FBS。这可以防止无意的的通道表达可能产生的四环素污染的血清。诱导珥通道表达只在一个包括1微克/毫升四环素的每个克隆重复的井。重复将作为未诱导以及“背景”的控制。执行TL +通量检测下。 </li></ol> 3。一般TL +通量检测过程<ol><li>铊+通量实验的前一天，游离细胞，定性和定量分析的细胞悬浮液的密度为2.1和2.2节中描述的。板20000单克隆细胞稳定转染用珥通道基因的BD PureCoat胺涂覆用Thermo多点Combi机或一个多通道移液器的384孔板的每个孔中的兴趣。使用BH培养基含有10％透析FBS用于电镀。注意，一些细胞将培养过夜，用1微克/毫升四环素诱导珥通道表达，而另一些则不会。诱导和未诱导的细胞的位置会有所不同，为每个类型的实验，并在图1中所示。 </li><li>第二天，在显微镜下检查板，以确保细胞是贴壁，并均匀地分布在整个底部孔中。该井应该是80％至90％汇合。 </li><li>使用一个ELx405洗板机更换细胞培养基中与20μl每孔的HBSS测定缓冲液中含有20mM HEPES缓冲至pH为7.3，用NaOH。另外，人们也可以使用“轻拂满贯”方法，其中，所述板反转并抢购急剧下降到废物容器中喷射介质，然后拍拍堆叠纸巾上，以除去剩余的媒体。立即重新添加HBSS测定缓冲液中的板，以防止在细胞从干燥剂。 </li><li>准备FluoZin-2，AM染料加载溶液。短暂离心管后，溶解于100μl的无水DMSO中的50微克的FluoZin-2粉末。此时，1,000×储备溶液的等分试样在-20℃下可存储供以后使用。准备使用时，加入50μL20％的重量每的量普朗尼克F-127/DMSO解决方案的染料和混合轻轻移液。一次，不要冷冻染料聚醚F-127已经补充说，低的温度可能会导致表面活性剂的溶液中沉淀出来。加入150微升体积的FluoZin-2，HBSS的AM/Pluronic-F127至100毫升的试验缓冲液中，并轻轻地混合，使染料的上样缓冲液。使用多点Combi机或者多通道移液器，染料加样缓冲液中加入20μl的每个孔中已经含有20微升测定的HBSS缓冲液。细胞在室温下温育约1小时（通常孵育已经在黑暗中进行，虽然目前还没有直接的证据表明，它是必要的）。 </li><li>当细胞被加载染料，准备了TL +刺激板。新鲜的0.5克碳酸氢钠溶解在50毫升的5x铊+刺激缓冲液含有1mM的硫酸镁，1.8 mM的硫酸钙，5mM葡萄糖，10mM HEPES和12毫铊+干燥。另外，碳酸氢钠可以取代葡萄糖酸钠，如果，例如，溶液的pH值必须保持在很窄的范围和二氧化碳损失是一个问题。盖上管盖紧紧地限制二氧化碳的逸出，并倒转数次，直到进入溶液碳酸氢钠。使用多通道移液器，聚丙烯384孔板中（ 表1）的各孔中的溶液中加入50μl。 </li><li>后，细胞已被加载与FluoZin-2，AM，清洗板与ELx405洗板机，或使用“轻拂满贯”的方法，如上述3.3节中描述的。根据不同类型的Tl +通量以进行实验，重新添加的HBSS在各孔的测定缓冲液20微升或40微升。现在已经准备好为实验板。 </li><li>细胞和Tl +板成一个滨松功能的药物筛选系统（FDSS）或等效动力学成像板集成的液体分配能力的读者。基于荧光素/荧光染料，如FLUO-4使用的适当的过滤器。 </li><li>设置添加一个单一的协议，使10μL的5倍TL +series已添加到相应的单元板极的孔中含有40微升的HBSS测定缓冲液。记录基线荧光以1 Hz的采样频率为至少10秒。该井的井荧光应该是统一的，稳定的，最佳的细胞电镀，清洗，染料负载条件下建立后，整个板。一个集成的384通道移液器是用来同时添加的Tl +刺激缓冲液，每孔。 </li><li>记录至少2分钟，使捕获的Tl +诱导的荧光增加的速率和峰值用于离线分析。 </li></ol> 4。最佳TL +浓度的测定<ol><li> +铊容易渗透到最内向整流K +通道。为了确保表明Tl +浓度不超过动态范围的Tl +报告染料FluoZin-2，1应该凭经验确定要使用的最佳的铊+浓度在高-叔hroughput屏幕上。我们推荐的TL +浓度，唤起通道被最大程度地激活的条件下，最大荧光反应（EC 80）的80％。 </li><li>板，染料负载和洗涤细胞，如上述第3节，留下的HBSS测定缓冲液中，在每个孔中加入40μl在BD PureCoat胺涂层或等效的聚-D-赖氨酸涂覆的384孔板。所示，在图1A中，第1列和行的A1-A23应当包含尚未四环素诱导，因此不表达珥感兴趣通道的细胞的板图。 FluoZin-2荧光信号，将被用于从非诱导井的高低来决定的Tl +通量通过内源性途径，如的Na + - K +-ATP酶泵和电压门控钾通道，该通道通常表示在HEK- 293细胞。 </li><li>使用安捷伦Bravo自动化液体处理平台或手动移液准备11点TL +浓度系列稀释在HBSS分析缓冲液中。通常情况下，3倍稀释系列，范围从100％到0.002％Tl十是在测定中评价。该系列应浓度的5倍，因为TL +解决方案将稀释1:5，在最后检测。准备的稀释系列稀释的标准5倍铊+第3节中描述的缓冲液，与5倍铊+无缓冲液含有1mM的硫酸镁，1.8 mM的硫酸钙，5mM葡萄糖和10mM HEPES。同样，新鲜溶解0.5克碳酸氢钠，50毫升的最终铊+缓冲电镀前立即在聚丙烯384 -孔板中，根据在图2A中所示的板图。 </li><li>将细胞刺激和Tl +板到FDSS。设置一个单一的附加 ​​组件的协议，使10微升的5倍的铊+系列被添加到相应的孔中含有40微升的HBS的单元板极小号测定缓冲液。记录基线FluoZin-2型荧光，持续10秒，然后再添加铊+的板。重复3天的实验，建立了重复性的结果。 </li></ol> 5。测定的检测灵敏度，以DMSO <ol><li>审问在高吞吐量的屏幕的小分子溶解在有机溶剂的二甲亚砜（DMSO）中，它本身可以影响性能的测定。因此，必须首先进行检查的检测的灵敏度对DMSO建立屏幕中允许的最高的DMSO浓度。 </li><li>板，染料负载和洗BD PureCoat胺涂层的384孔板中的细胞中，如上述第3条，离开各孔中的20μl的HBSS中的试验缓冲液。整个板块应该包含细胞被诱导与四环素（ 图3A）。 </li><li>使用一个Bravo自动化液体处理平台或手动移液准备11点DMSO系列稀释在HBSS分析缓冲。通常情况下，范围从10％至0.01％体积/体积DMSO 2 - 倍稀释系列评价在测定中的活性。该系列产品2倍的浓度应在，因为DMSO的解决方案将被摊薄一半在最后的分析。应镀的稀释系列的聚丙烯384 -孔板中，根据图3A中所示的板图。 </li><li>准备了5倍的TL +刺激EC 80或最佳TL +浓度在第4节中确定的基础上板。 </li><li> FDSS进入细胞，DMSO和Tl +刺激板装入。设置了两个附加的协议，以便添加到单元板极的相应的孔中含有20μl的HBSS中测定缓冲液20微升的2倍浓度的DMSO系列。发表DMSO为相同的时间量，在一个屏幕上，将细胞暴露于小分子的车辆上的细胞。记录基线FluoZin-2型荧光在至少10秒，然后向每孔加入10μl的5x铊+缓冲单元板极。重复3天的实验，建立了重复性的结果。 </li><li>该试验应该是耐DMSO的浓度至少为0.1％体积/体积，对于一个典型的高通量的屏幕，在该屏幕中的测试化合物在浓度为10μM。 </li></ol> 6。已知的药理调制器的灵敏度测定<ol><li>确定最优的铊+浓度和DMSO公差的测定后，已知的影响珥通道介导的铊+流的药理活性剂对应该进行检查。这一系列试验将评估了检测的灵敏度，能力排名，为了化合物，根据其效力，能够到分类，根据他们的模式的有效性（ 例如，激活剂，抑制剂）化合物，并确定乖巧的控制化合物到可以使用后面的分析其在才有发展和屏幕。 </li><li>板，染料负载和洗BD PureCoat胺涂层的384孔板中的细胞中，如上述第3条，离开各孔中的20μl的HBSS中的试验缓冲液。第1列和行的A1-A23应包含细胞没有被诱导用四环素（ 图4A）。 </li><li>使用一个Bravo自动化液体处理平台或手动移液，已知调制器在HBSS分析缓冲准备11点的浓度稀释系列。通常情况下，范围从100μM2 nM的3倍稀释系列评价在测定中的活性。该系列产品2倍的浓度应在，因为DMSO的解决方案将被摊薄一半在最后的分析。稀释系列应被镀在一式三份的聚丙烯384 -孔板中，根据图4A中所示的板图。成为确保稀释，使得最终DMSO的浓度是相同的药物治疗和减塔之间<html> n或等于第5节中定义的最大允许的DMSO浓度。 </li><li>准备了5倍TL +刺激TL +的最佳浓度确定在第4节的基础上板。 </li><li>装入电池，化合物和Tl +刺激板块到FDSS。设置了两个附加的协议，因此，20微升的2倍浓度的化合物series已添加到相应的单元板极的孔中含有20μl的HBSS中测定缓冲液。添加的化合物的单元板极和最多孵育20分钟。请注意，可以通过运行在几个不同的孵育时间选择了一系列的实验来确定最佳孵育时间的化合物的检测到最佳的信号背景比。记录基线FluoZin-2荧光为至少10秒的单元板极的各孔中加入10微升的5x铊+缓冲之前。重复3天的实验，建立了重复性的结果。 </li></ol>_title“棋盘分析&gt; 7。 <ol><li>的均匀性和再现性的测定将在接下来的一系列的实验中，使用“棋盘”分析评价。通常情况下，一个控制抑制剂镀在每一个孔的每列和每行的384孔板的其它，如在图5A中示出。要严格评估中的噪声检测，EC 80的抑制剂浓度应该被使用。加入DMSO作为车辆控制的其它井。 TL +流在DMSO和使用药物处理的细胞的Z总理，到井井的两个种群的变异性，统计方法，计算出一个数值。 Ž总理是使用以下公式计算： 于黄金= 1 - （3SD P + 3SD）/平均 p +意味着Ň| 其中SD为标准偏差，p是不羁通量和n被完全抑制磁通值。分析与Z&#39;值大于或等于0.5的3个独立的天被认为是小号高通量筛选uitable为。 </li><li>板，染料负载和洗BD PureCoat胺涂层的384孔板中的细胞中，如上述第3条，离开各孔中的20μl的HBSS中的试验缓冲液。需要注意的是，整个板块应与四环素引起的。 </li><li>通过移液到384孔聚丙烯板，使用多通道移液器，车型化合物/ DMSO板的80微升/孔的一个已知的抑制剂在测定的浓度在6.5节中的目标珥通道，和0.1％体积/体积DMSO如在图5A中所示的车辆。添加已知的抑制剂，以及A1开始使用多通道移液器和备用以及B2等，以及B24。重复此过程，DMSO以及B1等以及A24。板的最终布局应符合的棋盘。 </li><li>准备了5倍TL +刺激板的基础上的最佳TL +在第4节中确定。 </li><li>装入电池，化合物和TL +刺激板块到FDSS。设置了两个附加的协议，因此，20微升的2倍浓度的化合物series已添加到相应的单元板极的孔中含有20μl的HBSS中测定缓冲液。添加的化合物的单元板极和最多20分钟，在室温下孵育。记录基线FluoZin-2荧光为至少10秒的单元板极的各孔中加入10微升的5x铊+缓冲之前。重复3天的实验，建立了重复性的结果。 </li></ol> 8。试点屏幕<ol><li>在检测方法开发的最后阶段，几千元的化合物进行试点屏幕最终将被用于在大型高通量筛选的条件下检测的性能进行评估。 </li><li>板，染料负载和洗涤细胞BD PureCoat胺涂层的384孔板中，如上述第3节中描述的），留下在各孔中的20μl的HBSS中的测定缓冲液。请注意，整个板块应培养过夜，与四环素诱导珥通道表达。 </li><li>选择约2000至3000的结构不同的化合物以进行测试。它往往是适当和方便使用的化合物与已知的活动可能富集离子通道调节集合。这些收藏包括光谱采集（MICROSOURCE）和LOPAC的集合（Sigma公司）。此外，谨慎的做法是已知调制器的KIR的兴趣在试点屏幕。理想情况下，这些化合物将被添加到以双盲的方式，以允许命中采摘后述的方法的比较测试的井。准备的化合物在384孔聚丙烯板（目标板）使用Labcyte公司回音的液体处理器或合适的销工具从MLPCN集合（源板）中选择的化合物在DMSO中的适当体积的转移目的地板需要注意的是试验化合物是只有在列3-22;在每一个其他孔中的列1，2，23，24中添加已知的抑制剂在已知的浓度完全抑制，如在第7条中确定珥。在第1列，2，23，和24的剩余的孔中加适当体积的DMSO产生DMSO浓度匹配的车辆的控制。井检测缓冲液使用多点的Combi冲淡一切。通常情况下，将试验化合物20μM，其目标筛选浓度的2倍以上。 </li><li>请TL +刺激TL +的最佳浓度确定在第4节的基础上板。 </li><li>执行试点屏幕。请注意错开协议的执行步骤保持一致的板之间的时间运行在试点筛选。 </li><li>一旦试点屏幕完成后，从第1列，第2，23和24使用的Z贷方程分析的棋盘井。检查板，显示黄金Z-值小于0.5，以确定较差的分离控制人口的来源。点击可以ë选择使用了一些方法。对于导频的屏幕，它是共同的计算的平均值和标准偏差的车辆控制人口，并挑选根据井生产&gt; / = 3的车辆控制的平均值的标准偏差的值的命中。 </li><li>命中采摘，复试选定的命中，一式两份，每份2套牌。板的一组包含的稳定珥四环素和其他包含珥的稳定细胞系在存在四环素的情况下，单元格中。验证次数复检阳性复检的两个板中的至少一个，并可以考虑不显示显着的活性，在非诱导板的命中。检查验证的暗杀名单，以确定有多少“隐藏”控制样品进行检测。没有检测到的控制在试点屏幕显示需要进一步筛选或命中采摘参数的优化。 </li></ol>

<ol>
	<li>Ehrlich, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inward+rectifier+potassium+currents+as+a+target+for+atrial+fibrillation+therapy.">Inward rectifier potassium currents as a target for atrial fibrillation therapy.</a> J. Cardiovasc. Pharmacol. 52 (2), 129 (2008).</li><li>Bhave, G., Lonergan, D., Chauder, B. A., Denton, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Small-molecule+modulators+of+inward+rectifier+K++channels:+recent+advances+and+future+possibilities.">Small-molecule modulators of inward rectifier K+ channels: recent advances and future possibilities.</a> Future Med. Chem. 2 (5), 757 (2010).</li><li>Swartz, K. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tarantula+toxins+interacting+with+voltage+sensors+in+potassium+channels.">Tarantula toxins interacting with voltage sensors in potassium channels.</a> Toxicon. 49 (2), 213 (2007).</li><li>Jin, W., Lu, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+high-affinity+inhibitor+for+inward-rectifier+K++channels.">A novel high-affinity inhibitor for inward-rectifier K+ channels.</a> Biochemistry. 37 (38), 13291 (1998).</li><li>Jin, W., Lu, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthesis+of+a+stable+form+of+tertiapin:+a+high-affinity+inhibitor+for+inward-rectifier+K++channels.">Synthesis of a stable form of tertiapin: a high-affinity inhibitor for inward-rectifier K+ channels.</a> Biochemistry. 38 (43), 14286 (1999).</li><li>Roy, A., McDonald, P. R., Sittampalam, S., Chaguturu, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Open+access+high+throughput+drug+discovery+in+the+public+domain:+a+Mount+Everest+in+the+making.">Open access high throughput drug discovery in the public domain: a Mount Everest in the making.</a> Curr. Pharm. Biotechnol. 11 (7), 764 (2010).</li><li>Weaver, C. 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=A+thallium-sensitive,+fluorescence-based+assay+for+detecting+and+characterizing+potassium+channel+modulators+in+mammalian+cells.">A thallium-sensitive, fluorescence-based assay for detecting and characterizing potassium channel modulators in mammalian cells.</a> J. Biomol. Screen. 9 (8), 671 (2004).</li><li>Lewis, L. M., 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=High-throughput+screening+reveals+a+small-molecule+inhibitor+of+the+renal+outer+medullary+potassium+channel+and.">High-throughput screening reveals a small-molecule inhibitor of the renal outer medullary potassium channel and.</a> 76 (5), 1094 (2009).</li><li>Bhave, G., 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=Development+of+a+selective+small-molecule+inhibitor+of+Kir1.1,+the+renal+outer+medullary+potassium+channel.">Development of a selective small-molecule inhibitor of Kir1.1, the renal outer medullary potassium channel.</a> Mol. Pharmacol. 79 (1), 42 (2011).</li><li>Raphemot, R., 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=Discovery,+characterization,+and+structure-activity+relationships+of+an+inhibitor+of+inward+rectifier+potassium+(Kir)+channels+with+preference+for+Kir2.3,+Kir3.x,+and+Kir7.1.">Discovery, characterization, and structure-activity relationships of an inhibitor of inward rectifier potassium (Kir) channels with preference for Kir2.3, Kir3.x, and Kir7.1.</a> Front Pharmacol. 2, 75 (2012).</li><li>Niswender, C. M., 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=A+novel+assay+of+Gi/o-linked+G+protein-coupled+receptor+coupling+to+potassium+channels+provides+new+insights+into+the+pharmacology+of+the+group+III+metabotropic+glutamate+receptors.">A novel assay of Gi/o-linked G protein-coupled receptor coupling to potassium channels provides new insights into the pharmacology of the group III metabotropic glutamate receptors.</a> Mol. Pharmacol. 73 (4), 1213 (2008).</li></ol>

没有利益冲突的声明。

数据处理：一旦数据被收集，在分析中的一个共同的步骤涉及到的各孔的荧光响应到其初始值，在开始的实验中，F 0，F，正常化。这通常被称为“静态比”和符号“F / F 0”。静态比操作将在F 0的情况下，主要是由指示剂染料基本上纠正许多因素，如照明，信号采集disuniformities，和细胞数。染料信号弱的情况下，或在系统中的的背景荧光或反射高，静...

An erratum was issued for: <a href="https://www.jove.com/video/4209/high-throughput-screening-for-small-molecule-modulators-inward">High-throughput Screening for Small-molecule Modulators of Inward Rectifier Potassium Channels</a>. The Protocol section has been updated.
The formula in step 7.1 of the Protocol has been updated from:
Z prime = 1- (3SDp + 3SDn)/|meanp + meann |
to:
Z prime = 1- (3SDp + 3SDn)/|meanp - meann |

An erratum was issued for: <a href="https://www.jove.com/video/4209/high-throughput-screening-for-small-molecule-modulators-inward">High-throughput Screening for Small-molecule Modulators of Inward Rectifier Potassium Channels</a>. The Protocol section has been updated.

Erratum: High-throughput Screening for Small-molecule Modulators of Inward Rectifier Potassium Channels

<table border="1"><tbody><tr><td> 的试剂的名称 </td><td> 公司 </td><td> 目录号 </td><td> 评论 </td></tr><tr><td> pcDNA5/TO </td><td> Invitrogen公司</td><td> V1033-20 </td><td>四环素诱导表达载体</td></tr><tr><td> T-REX-HEK293细胞</td><td> Invitrogen公司</td><td> R71007 </td><td>四环素诱导的细胞系</td></tr><tr><td>脂质体LTX /加试剂</td><td> Invitrogen公司</td><td> 15338100 </td><td>转染试剂</td></tr><tr><td> FBS </td><td>亚特兰大生物</td><td> S11550 </td><td>细胞培养基</td></tr><tr><td> DMEM </td><td> Invitrogen公司</td><td> 11965 </td><td>细胞培养基</td></tr><tr><td>潮霉素B </td><td> Invitrogen公司</td><td> 10687-010 </td><td>细胞培养基</td></tr><tr><td>杀稻瘟小号</td><td> Invitrogen公司</td&gt; <td> R210-01 </td><td>细胞培养基</td></tr><tr><td>青霉素/链霉素</td><td> Invitrogen公司</td><td> 15140 </td><td>细胞培养基</td></tr><tr><td> HBSS价免费</td><td>敏达</td><td> 21022CV </td><td>细胞洗涤</td></tr><tr><td>胰蛋白酶-0.25％ </td><td>敏达</td><td> 25053CI </td><td>细胞分离</td></tr><tr><td>四环素盐酸</td><td>西格玛</td><td> T9823 </td><td>感应试剂</td></tr><tr><td>透析FBS </td><td>亚特兰大生物</td><td> S12650 </td><td>电镀媒体</td></tr><tr><td> FluoZin-2 </td><td> Invitrogen公司</td><td> F24189 </td><td>荧光染料</td></tr><tr><td>聚醚F-127 </td><td> Invitrogen公司</td><td> P-3000MP </td><td>染料吸附量</td></tr><tr><td> HBSS </td><td> Invitrogen公司</td><td> 14175 </td><td>分析缓冲液</td></tr><tr><td> HEPES </td><td> Invitrogen公司</td><td> 15630 </td><td>分析缓冲液</td></tr><tr><td> 碳酸氢钠 </td><td>西格玛</td><td> S6297 </td><td> TL +刺激缓冲</td></tr><tr><td> 硫酸镁 </td><td>西格玛</td><td> M2643 </td><td> TL +刺激缓冲</td></tr><tr><td>硫酸钙4·2H 2 O </td><td>西格玛</td><td> C3771 </td><td> TL +刺激缓冲</td></tr><tr><td> D-葡萄糖</td><td>西格玛</td><td> G7528 </td><td> TL +刺激缓冲</td></tr><tr><td>硫酸铊</td><td> Aldrich公司</td><td> 204625 </td><td> TL +刺激缓冲</td></tr><tr><td> HEPES </td><td>西格玛</td><td> H4034 </td><td> TL +刺激缓冲</td></tr><tr><td> DMSO </td><td>西格玛</td><td> D4540 </td><td>溶剂</td></tr><tr><td>八通道电子移液器</td><td>百得</td><td> E300 </td><td>在384细胞电镀 -孔板</td></tr><tr><td> BD PureCoat胺涂层的384孔板</td><td> BD Biosciences公司</td><td> 356719 </td><td>检测微孔板</td></tr><tr><td>回声合格384孔微孔板聚丙烯（384PP） </td><td> Labcyte公司</td><td> P-05525 </td><td>复合源微孔板</td></tr><tr><td> 384孔聚丙烯微孔板</td><td>格雷纳生物之一</td><td> 781280 </td><td></td></tr><tr><td>多点COMBI试剂分配器</td><td> Thermo Scientific的</td><td> 5840300 </td><td></td></tr><tr><td> ELx405洗板机</td><td>百特</td><td> ELx405HT </td><td>自动细胞清洗</td></tr><tr><td>回声液体处理</td><td> Labcyte公司</td><td> Labcyte公司回声550 </td><td></td></tr><tr><td> Bravo自动化液体处理平台</td><td>安捷伦科技公司</td><td>标准模型</td><td></td></tr><tr><td> HamamaTSU FDSS 6000 </td><td>滨松</td><td></td><td>动力学成像板读卡器</td></tr></tbody></table> 表1中 。材料与试剂名单。

high-throughput screening for small-molecule modulators of inward rectifier potassium channels

具体内向整流钾通道（基里巴斯）家庭成员的各种疾病，包括高血压，房颤，疼痛1,2推测的药物靶标。然而，在大多数情况下，对理解他们的治疗潜力甚至连最基本的生理功能方面取得的进展已经放缓缺乏良好的药理工具。事实上，分 ​​子药理学的内向整流家庭的已经远远落后的S4家族的电压门控钾通道（KV），其中一些已发现的的纳摩尔亲和力和高度选择性的肽毒素调节3。蜂毒的毒素tertiapin和其衍生物是有效的抑制剂的Kir1.1的Kir3通道4,5，但肽是使用有限的治疗以及实验由于它们的抗原特性和生物利用度差，代谢稳定性和组织外显率。强效的发展改进的药理特性和选择性的小分子探针将是一个关键Kir通道的生理和治疗潜力的充分认识。 分子库探针生产中心网（MLPCN）的支持由美国国立卫生研究院（NIH）共同基金创造了机会学术界的科学家开始探测发现运动的分子靶点和信号转导通路需要更好的药理学6。 MLPCN提供研究获得行业规模筛选中心，药物化学和信息支持开发小分子探针，以阐明基因和基因网络的功能。关键的一步在进入到MLPCN是一个强大的目标或途径的具体分析，是适合高通量筛选（HTS）的发展。 在这里，我们介绍了如何开发基于荧光的铊（TL +）通量阿萨y的高通量化合物筛选7,8,9,10珥通道功能。该测定是基于的通透性上的K +通道孔的K +同源物铊+。市售的荧光铊+报告染料是用来检测通过孔隙的Tl +跨膜通量。有至少三个市售染料适合铊+磁通检测：BTC，FluoZin-2，7,8 FluxOR。该协议描述试验发展，使用FluoZin-2。虽然最初开发和销售锌指标，FluoZin-2是一个强大的和剂量依赖性增加，荧光发射后，TL +结合。我们开始工作前与FluoZin-2 FluxOR是7,8，并继续这样做9,10。然而，分析发展中的步骤基本上是相同的所有三种染料，用户应确定染料是最适合其特定的N电火工品。我们还讨论了检测的性能基准测试，必须达到被认为是进入到MLPCN的。由于铊+容易渗透K +通道，该法应适用于K +通道的目标。

四环素诱导表达系统的使用提供了一个方便的内部控制，用于区分铊+通量通过内源性途径和珥感兴趣通道的图1示出了一些实施例的不同类型的实验中使用的细胞接种地图。含有未诱导或四环素诱导的细胞的孔的位置用不同的颜色表示图2A显示了使用，以确定最佳的铊+浓度的检测开发和筛选化合物的源板图。渐变的颜色表示的3倍稀释系列，范围从100％到0....

Watch this Scientific Journal Video about High-throughput Screening for Small-molecule Modulators of Inward Rectifier Potassium Channels at JoVE.com

High-throughput Screening for Small-molecule Modulators of Inward Rectifier Potassium Channels

开发和验证测量活动的高通量化合物筛选的内向整流钾通道（基里巴斯）荧光定量检测方法。

内向整流钾通道的小分子调节剂的高通量筛选

Specific members of the inward rectifier potassium (Kir) channel family are postulated drug targets for a variety of disorders, including hypertension, ...

high-throughput-screening-for-small-molecule-modulators-inward

Research

JoVE Journal

Biology

14.9K 次观看.  Vanderbilt University School of Medicine. 开发和验证测量活动的高通量化合物筛选的内向整流钾通道（基里巴斯）荧光定量检测方法。

视频: High-throughput Screening for Small-molecule Modulators of Inward Rectifier Potassium Channels

High-throughput Screening and Biosensing with Fluorescent C. elegans Strains

High-throughput screening (HTS) is a powerful approach for identifying chemical modulators of biological processes. However, many compounds identified in screens using cell culture models are often found to be toxic or pharmacologically inactive in vivo1-2. Screening in whole animal models can help avoid these pitfalls and streamline the path to drug development.
C. elegans is a multicellular model organism well suited for HTS. It is small (&lt;1 mm) and can be economically cultured and dispensed in liquids. C. elegans is also one of the most experimentally tractable animal models permitting rapid and detailed identification of drug mode-of-action3.
We describe a protocol for culturing and dispensing fluorescent strains of C. elegans for high-throughput screening of chemical libraries or detection of environmental contaminants that alter the expression of a specific gene. Large numbers of developmentally synchronized worms are grown in liquid culture, harvested, washed, and suspended at a defined density. Worms are then added to black, flat-bottomed 384-well plates using a peristaltic liquid dispenser. Small molecules from a chemical library or test samples (e.g., water, food, or soil) can be added to wells with worms. In vivo, real-time fluorescence intensity is measured with a fluorescence microplate reader. This method can be adapted to any inducible gene in C. elegans for which a suitable reporter is available. Many inducible stress and developmental transcriptional pathways are well defined in C. elegans and GFP transgenic reporter strains already exist for many of them4. When combined with the appropriate transgenic reporters, our method can be used to screen for pathway modulators or to develop robust biosensor assays for environmental contaminants. 
We demonstrate our C. elegans culture and dispensing protocol with an HTS assay we developed to monitor the C. elegans cap &#x2018;n&#x2019; collar transcription factor SKN-1. SKN-1 and its mammalian homologue Nrf2 activate cytoprotective genes during oxidative and xenobiotic stress5-10. Nrf2 protects mammals from numerous age-related disorders such as cancer, neurodegeneration, and chronic inflammation and has become a major chemotherapeutic target11-13.Our assay is based on a GFP transgenic reporter for the SKN-1 target gene gst-414, which encodes a glutathione-s transferase6. The gst-4 reporter is also a biosensor for xenobiotic and oxidative chemicals that activate SKN-1 and can be used to detect low levels of contaminants such as acrylamide and methyl-mercury15-16.

High-throughput Screening and Biosensing with Fluorescent C. elegans Strains

A procedure for liquid-based culturing and dispensing of C. elegans strains expressing fluorescent reporter proteins is described that does not require expensive sorting equipment. This approach can be applied to numerous inducible C. elegans genes for drug discovery or biosensing of contaminants.

高通量筛选和生物传感与荧光 C。线虫菌株

High-throughput screening (HTS) is a powerful approach for identifying chemical modulators of biological processes.  However, many compounds identified in ...

科研

生物学

High Throughput Screening of Fungal Endoglucanase Activity in Escherichia coli

Cellulase enzymes (endoglucanases, cellobiohydrolases, and &beta;-glucosidases) hydrolyze cellulose into component sugars, which in turn can be converted into fuel alcohols1. The potential for enzymatic hydrolysis of cellulosic biomass to provide renewable energy has intensified efforts to engineer cellulases for economical fuel production2. Of particular interest are fungal cellulases3-8, which are already being used industrially for foods and textiles processing.
Identifying active variants among a library of mutant cellulases is critical to the engineering process; active mutants can be further tested for improved properties and/or subjected to additional mutagenesis. Efficient engineering of fungal cellulases has been hampered by a lack of genetic tools for native organisms and by difficulties in expressing the enzymes in heterologous hosts. Recently, Morikawa and coworkers developed a method for expressing in E. coli the catalytic domains of endoglucanases from H. jecorina3,9, an important industrial fungus with the capacity to secrete cellulases in large quantities. Functional E. coli expression has also been reported for cellulases from other fungi, including Macrophomina phaseolina10 and Phanerochaete chrysosporium11-12. 
We present a method for high throughput screening of fungal endoglucanase activity in E. coli. (Fig 1) This method uses the common microbial dye Congo Red (CR) to visualize enzymatic degradation of carboxymethyl cellulose (CMC) by cells growing on solid medium. The activity assay requires inexpensive reagents, minimal manipulation, and gives unambiguous results as zones of degradation (&#x201C;halos&#x201D;) at the colony site. Although a quantitative measure of enzymatic activity cannot be determined by this method, we have found that halo size correlates with total enzymatic activity in the cell. Further characterization of individual positive clones will determine , relative protein fitness. 
Traditional bacterial whole cell CMC/CR activity assays13 involve pouring agar containing CMC onto colonies, which is subject to cross-contamination, or incubating cultures in CMC agar wells, which is less amenable to large-scale experimentation. Here we report an improved protocol that modifies existing wash methods14 for cellulase activity: cells grown on CMC agar plates are removed prior to CR staining. Our protocol significantly reduces cross-contamination and is highly scalable, allowing the rapid screening of thousands of clones. In addition to H. jecorina enzymes, we have expressed and screened endoglucanase variants from the Thermoascus aurantiacus and Penicillium decumbens (shown in Figure 2), suggesting that this protocol is applicable to enzymes from a range of organisms.

High Throughput Screening of Fungal Endoglucanase Activity in Escherichia coli

We describe a low cost, high throughput method to screen for fungal endoglucanase activity in E. coli. The method relies on a simple visual readout of substrate degradation, does not require enzyme purification, and is highly scalable. This allows for the rapid screening of large libraries of enzyme variants.

真菌内切葡聚糖酶的活性高通量筛选大肠埃希氏菌</em

Cellulase enzymes (endoglucanases, cellobiohydrolases, and &beta;-glucosidases) hydrolyze cellulose into component sugars, which in turn can be converted ...

A Fluorescent Screening Assay for Identifying Modulators of GIRK Channels

G protein-gated inward rectifier K+ (GIRK) channels function as cellular mediators of a wide range of hormones and neurotransmitters and are expressed in the brain, heart, skeletal muscle and endocrine tissue1,2. GIRK channels become activated following the binding of ligands (neurotransmitters, hormones, drugs, etc.) to their plasma membrane-bound, G protein-coupled receptors (GPCRs). This binding causes the stimulation of G proteins (Gi and Go) which subsequently bind to and activate the GIRK channel. Once opened the GIRK channel allows the movement of K+ out of the cell causing the resting membrane potential to become more negative. As a consequence, GIRK channel activation in neurons decreases spontaneous action potential formation and inhibits the release of excitatory neurotransmitters. In the heart, activation of the GIRK channel inhibits pacemaker activity thereby slowing the heart rate.
GIRK channels represent novel targets for the development of new therapeutic agents for the treatment neuropathic pain, drug addiction, cardiac arrhythmias and other disorders3. However, the pharmacology of these channels remains largely unexplored. Although a number of drugs including anti-arrhythmic agents, antipsychotic drugs and antidepressants block the GIRK channel, this inhibition is not selective and occurs at relatively high drug concentrations3.
Here, we describe a real-time screening assay for identifying new modulators of GIRK channels. In this assay, neuronal AtT20 cells, expressing GIRK channels, are loaded with membrane potential-sensitive fluorescent dyes such as bis-(1,3-dibutylbarbituric acid) trimethine oxonol [DiBAC4(3)] or HLB 021-152 (Figure 1). The dye molecules become strongly fluorescent following uptake into the cells (Figure 1). Treatment of the cells with GPCR ligands stimulates the GIRK channels to open. The resulting K+ efflux out of the cell causes the membrane potential to become more negative and the fluorescent signal to decrease (Figure 1). Thus, drugs that modulate K+ efflux through the GIRK channel can be assayed using a fluorescent plate reader. Unlike other ion channel screening assays, such atomic absorption spectrometry4 or radiotracer analysis5, the GIRK channel fluorescent assay provides a fast, real-time and inexpensive screening procedure.

A real-time screening procedure for identifying drugs that interact with G protein-gated inward rectifier K+ (GIRK) channels is described. The assay utilizes membrane potential-sensitive fluorescent dyes to measure GIRK channel activity. This technique is adaptable for use on a number of cell lines.

一个荧光识别GIRK通道调制器的筛选试验

G protein-gated inward rectifier K+ (GIRK) channels function as cellular mediators of a wide range of hormones and neurotransmitters and are expressed in ...

High-throughput Functional Screening using a Homemade Dual-glow Luciferase Assay

We present a rapid and inexpensive high-throughput screening protocol to identify transcriptional regulators of alpha-synuclein, a gene associated with Parkinson&#39;s disease. 293T cells are transiently transfected with plasmids from an arrayed ORF expression library, together with luciferase reporter plasmids, in a one-gene-per-well microplate format. Firefly luciferase activity is assayed after 48 hr to determine the effects of each library gene upon alpha-synuclein transcription, normalized to expression from an internal control construct (a hCMV promoter directing Renilla luciferase). This protocol is facilitated by a bench-top robot enclosed in a biosafety cabinet, which performs aseptic liquid handling in 96-well format. Our automated transfection protocol is readily adaptable to high-throughput lentiviral library production or other functional screening protocols requiring triple-transfections of large numbers of unique library plasmids in conjunction with a common set of helper plasmids. We also present an inexpensive and validated alternative to commercially-available, dual luciferase reagents which employs PTC124, EDTA, and pyrophosphate to suppress firefly luciferase activity prior to measurement of Renilla luciferase. Using these methods, we screened 7,670 human genes and identified 68 regulators of alpha-synuclein. This protocol is easily modifiable to target other genes of interest.

We present a rapid and inexpensive screening method for identifying transcriptional regulators using high-throughput robotic transfections and a homemade dual-glow luciferase assay. This protocol rapidly generates direct side-by-side functional data for thousands of genes and is easily modifiable to target any gene of interest.

使用自制双辉光萤光素酶检测的高通量筛选功能

We present a rapid and inexpensive high-throughput screening protocol to identify transcriptional regulators of alpha-synuclein, a gene associated with ...

A Method of Permeabilization of Drosophila Embryos for Assays of Small Molecule Activity

The Drosophila embryo has long been a powerful laboratory model for elucidating molecular and genetic mechanisms that control development. The ease of genetic manipulations with this model has supplanted pharmacological approaches that are commonplace in other animal models and cell-based assays. Here we describe recent advances in a protocol that enables application of small molecules to the developing fruit fly embryo. The method details steps to overcome the impermeability of the eggshell while maintaining embryo viability. Eggshell permeabilization across a broad range of developmental stages is achieved by application of a previously described d-limonene embryo permeabilization solvent (EPS1) and by aging embryos at reduced temperature (18 &#176;C) prior to treatments. In addition, use of a far-red dye (CY5) as a permeabilization indicator is described, which is compatible with downstream applications involving standard red and green fluorescent dyes in live and fixed preparations. This protocol is applicable to studies using bioactive compounds to probe developmental mechanisms as well as for studies aimed at evaluating teratogenic or pharmacologic activity of uncharacterized small molecules.

A Method of Permeabilization of Drosophila Embryos for Assays of Small Molecule Activity

Introduction of small molecules to the developing Drosophila embryo offers great potential for characterizing biological activity of novel compounds, drugs, and toxins as well as for probing fundamental developmental pathways. Methods described herein outline steps that overcome natural barriers to this approach, expanding the utility of the Drosophila embryo model.

的透的一种方法<em&gt;果蝇</em&gt;胚胎用于小分子活性的测定

The Drosophila embryo has long been a powerful laboratory model for elucidating molecular and genetic mechanisms that control development. The ease of ...

A Manual Small Molecule Screen Approaching High-throughput Using Zebrafish Embryos

Zebrafish have become a widely used model organism to investigate the mechanisms that underlie developmental biology and to study human disease pathology due to their considerable degree of genetic conservation with humans. Chemical genetics entails testing the effect that small molecules have on a biological process and is becoming a popular translational research method to identify therapeutic compounds. Zebrafish are specifically appealing to use for chemical genetics because of their ability to produce large clutches of transparent embryos, which are externally fertilized. Furthermore, zebrafish embryos can be easily drug treated by the simple addition of a compound to the embryo media. Using whole-mount in situ hybridization (WISH), mRNA expression can be clearly visualized within zebrafish embryos. Together, using chemical genetics and WISH, the zebrafish becomes a potent whole organism context in which to determine the cellular and physiological effects of small molecules. Innovative advances have been made in technologies that utilize machine-based screening procedures, however for many labs such options are not accessible or remain cost-prohibitive. The protocol described here explains how to execute a manual high-throughput chemical genetic screen that requires basic resources and can be accomplished by a single individual or small team in an efficient period of time. Thus, this protocol provides a feasible strategy that can be implemented by research groups to perform chemical genetics in zebrafish, which can be useful for gaining fundamental insights into developmental processes, disease mechanisms, and to identify novel compounds and signaling pathways that have medically relevant applications.

The zebrafish is an excellent experimental organism to study vertebrate developmental processes and model human disease. Here, we describe a protocol on how to perform a manual high-throughput chemical screen in zebrafish embryos with a whole-mount in situ hybridization (WISH) read-out.

手册小分子屏幕接近高通量使用斑马鱼胚胎

Zebrafish have become a widely used model organism to investigate the mechanisms that underlie developmental biology and to study human disease pathology ...

Recovery of Adult Zebrafish Hearts for High-throughput Applications

Use of the zebrafish model system for studying development, regeneration, and disease is expanding toward use of adult hearts for cell dissociation and purification of RNA, DNA, and proteins. All of these applications demand the rapid recovery of significant numbers of zebrafish hearts to avoid gene regulatory, metabolic, and other changes that begin after death. Adult zebrafish hearts are also required for studying heart structure for a variety of mutants and for studying heart regeneration. However, the traditional zebrafish heart dissection is slow and difficult and requires specialized tools, making large-scale dissection of adult zebrafish hearts tedious. Traditional methods also harbor the risk of damaging the heart during the dissection. Here, we describe a method for dissection of adult zebrafish hearts that is fast, reproducible, and preserves heart architecture. Furthermore, this method does not require specialized tools, is painless for the zebrafish, can be performed on fresh or fixed specimens, and can be performed on zebrafish as young as one month old. The approach described expands the use of adult zebrafish for cardiovascular research.

Use of zebrafish for cardiovascular research is expanding towards research on adult hearts. For these applications, quick and simple isolation of cardiac tissues is key to avoid post-mortem changes and to obtain an adequate number of samples. Here, we describe a fast and reproducible method for dissecting adult zebrafish hearts.

恢复成年斑马鱼的心脏的高通量应用

Use of the zebrafish model system for studying development, regeneration, and disease is expanding toward use of adult hearts for cell dissociation and ...

High-throughput Screening for Chemical Modulators of Post-transcriptionally Regulated Genes

Both transcriptional and post-transcriptional regulation have a profound impact on genes expression. However, commonly adopted cell-based screening assays focus on transcriptional regulation, being essentially aimed at the identification of promoter-targeting molecules. As a result, post-transcriptional mechanisms are largely uncovered by gene expression targeted drug development. Here we describe a cell-based assay aimed at investigating the role of the 3&#39; untranslated region (3&#8217; UTR) in the modulation of the fate of its mRNA, and at identifying compounds able to modify it. The assay is based on the use of a luciferase reporter construct containing the 3&#8217; UTR of a gene of interest stably integrated into a disease-relevant cell line. The protocol is divided into two parts, with the initial focus on the primary screening aimed at the identification of molecules affecting luciferase activity after 24 hr of treatment. The second part of the protocol describes the counter-screening necessary to discriminate compounds modulating luciferase activity specifically through the 3&#8217; UTR. In addition to the detailed protocol and representative results, we provide important considerations about the assay development and the validation of the hit(s) on the endogenous target. The described cell-based reporter gene assay will allow scientists to identify molecules modulating protein levels via post-transcriptional mechanisms dependent on a 3&#8217; UTR.

Here we describe a cell-based reporter gene assay as a valuable tool to screen chemical libraries for compounds modulating post-transcriptional control mechanisms exerted through 3&#8217; UTR.

高通量筛选转录后调控基因的化学调节剂

Both transcriptional and post-transcriptional regulation have a profound impact on genes expression. However, commonly adopted cell-based screening assays ...

Identification of Kinase-substrate Pairs Using High Throughput Screening

We have developed a screening platform to identify dedicated human protein kinases for phosphorylated substrates which can be used to elucidate novel signal transduction pathways. Our approach features the use of a library of purified GST-tagged human protein kinases and a recombinant protein substrate of interest. We have used this technology to identify MAP/microtubule affinity-regulating kinase 2 (MARK2) as the kinase for a glucose-regulated site on CREB-Regulated Transcriptional Coactivator 2 (CRTC2), a protein required for beta cell proliferation, as well as the Axl family of tyrosine kinases as regulators of cell metastasis by phosphorylation of the adaptor protein ELMO. We describe this technology and discuss how it can help to establish a comprehensive map of how cells respond to environmental stimuli.

Protein phosphorylation is a central feature of how cells interpret and respond to information in their extracellular milieu. Here, we present a high throughput screening protocol using kinases purified from mammalian cells to rapidly identify kinases that phosphorylate a substrate(s) of interest.

激酶底物对鉴定使用高通量筛选


We have developed a screening platform to identify dedicated human protein kinases for phosphorylated substrates which can be used to elucidate novel ...

Automated Modular High Throughput Exopolysaccharide Screening Platform Coupled with Highly Sensitive Carbohydrate Fingerprint Analysis

Many microorganisms are capable of producing and secreting exopolysaccharides (EPS), which have important implications in medical fields, food applications or in the replacement of petro-based chemicals. We describe an analytical platform to be automated on a liquid handling system that allows the fast and reliable analysis of the type and the amount of EPS produced by microorganisms. It enables the user to identify novel natural microbial exopolysaccharide producers and to analyze the carbohydrate fingerprint of the corresponding polymers within one day in high-throughput (HT). Using this platform, strain collections as well as libraries of strain variants that might be obtained in engineering approaches can be screened. The platform has a modular setup, which allows a separation of the protocol into two major parts. First, there is an automated screening system, which combines different polysaccharide detection modules: a semi-quantitative analysis of viscosity formation via a centrifugation step, an analysis of polymer formation via alcohol precipitation and the determination of the total carbohydrate content via a phenol-sulfuric-acid transformation. Here, it is possible to screen up to 384 strains per run. The second part provides a detailed monosaccharide analysis for all the selected EPS producers identified in the first part by combining two essential modules: the analysis of the complete monomer composition via ultra-high performance liquid chromatography coupled with ultra violet and electrospray ionization ion trap detection (UHPLC-UV-ESI-MS) and the determination of pyruvate as a polymer substituent (presence of pyruvate ketal) via enzymatic oxidation that is coupled to a color formation. All the analytical modules of this screening platform can be combined in different ways and adjusted to individual requirements. Additionally, they can all be handled manually or performed with a liquid handling system. Thereby, the screening platform enables a huge flexibility in order to identify various EPS.

We present an automated modular high-throughput-method for the identification and characterization of microbial exopolysaccharides in small scale. This method combines a fast preselection to analyze the total amount of secreted polysaccharides with a detailed carbohydrate fingerprint to enable the fast screening of newly isolated bacterial strains or entire strain collections.