我们感谢工程和物理科学研究理事会（EPSRC）的资助（HLB）。此过程的拍摄过程中我们也感谢诗丽黛玮·阿雅潘Maalika Ramanoudjame对她的帮助。

类肽的1固相合成注:拟肽是使用固相拟肽合成的submonomer步骤手动合成。该方法允许高耦合效率和良好的最终产品的收率。上的固相合成也允许过量反应物可以在各步骤结束时容易地除去，并且该方法已在这里修改，以允许不同的官能化阳离子单体（ 即，精氨酸型和赖氨酸型残基）被包括在同一个序列1,2 <ol><li> 线性拟肽的合成 注意:在开始之前，合成开展安全评估。开展在通风橱中的所有反应，并穿戴适当的个人防护装备合适的（ 即一次性丁腈手套，防护眼镜和白大褂）。使用以下试剂和溶剂时要特别当心。二甲基甲酰胺（DMF）中是一种可疑的致畸以及dichloromethane（DCM）是一种致癌物。N，N&#39;-diisopropylcarbodiimide（DIC）和哌啶是通过呼吸道吸入危害眼睛，皮肤，并且可能会引起皮肤过敏。肼是一种可疑致癌物质，吸入致命，并导致严重灼伤皮肤或眼睛。溴乙酸也是危险的皮肤，眼睛和呼吸道，并可能导致在接触时灼伤。三氟乙酸（TFA）是一种挥发性液体，可引起严重烧伤这么小心处理。建议使用重型手套。 <ol><li> 0.12克Fmoc保护的Rink酰胺树脂（0.1毫摩尔，典型负载0.7毫摩尔/克）添加到封端20毫升聚丙烯反应容器中具有两个玻璃料。加入5毫升二甲基甲酰胺（DMF），以使树脂溶胀并离开该容器静置在室温下至少60分钟。采用固相萃取真空平台沥干DMF。 </li><li>向脱保护的溶胀树脂Fmoc基团，加入2毫升哌啶溶液（在DMF中20节％/ v）中。上放置容器在室温下（450转），并摇动器平台摇动5分钟。除去经由真空站中的溶液。 <ol><li>重复Fmoc去保护用2ml哌啶溶液中，摇动，在室温下15分钟。作为前沥干解决方案。 </li></ol></li><li>加入2毫升DMF并混合树脂30秒洗涤树脂。漏DMF中，并重复三次。 </li><li>对于乙酰化，加入1毫升溴乙酸溶液（0.6 M在DMF中）和0.2毫升N，N-&#39;-diisopropylcarbodiimide溶液（DIC，50％的DMF体积/体积）。离开反应容器摇动，在室温下20分钟。排出溶液，并用2毫升DMF三次洗涤树脂。 </li><li>对位移，加入1毫升的胺溶液（在DMF中的1.5M）。摇动，在室温下60分钟的树脂。排出溶液，并用2毫升DMF三次洗涤树脂。 <ol><li>要添加精氨酸型单体，按照步骤1.7。根据所需的拟肽序列，不同的胺将被添加。 </li></ol></li><li>重复步骤1.1.4和1.1.5。 </li><li>以包括胍官能单体（ 即 ，N-精氨酸），加入1 ml无保护的二胺溶液（在DMF中的1.5M），以树脂和动摇在室温下60分钟。 <ol><li>排出溶液，并用2毫升DMF三次洗涤树脂。 </li><li>添加2- acetyldimedone（0.2克，1毫摩尔以0.5毫升DMF，10当量）于DDE组添加到游离的伯胺和摇晃，在室温下60分钟。排出溶液，并用2毫升DMF三次洗涤树脂。 </li></ol></li><li>如在1.1.4至1.1.7直到所需序列是由继续submonomer合成。加入2毫升DMF洗涤树脂，并重复三次。 </li><li>去保护在树脂上的DDE基，加入4毫升2-％肼溶液（在DMF体积/体积）并摇动在室温下3分钟。排出溶液，并重复三次。 </li><li>排出溶液，并用2毫升DMF3吨洗涤树脂输入法。 </li><li>添加吡唑-1-甲脒（每游离胺6当量， 即每Ñ精氨酸单体，在DMF中的最小体积）和N，N-二异丙基乙胺或DIPEA（每游离胺6当量），并在室温下摇晃60分钟。 </li><li>排出溶液并用2ml二氯甲烷三次洗涤树脂。离开树脂在空气中干燥10分钟，然后将树脂可以存储直到切割（部分2）。 </li><li>暂停的合成，洗涤树脂用2毫升DMF三次。加入2毫升DMF，塞住合成容器并在通风橱中在室温下离开。 注:后（可形成除了第二移动一步，二酮哌嗪）的任何位移步骤合成可以暂停。 </li></ol></li><li> 侧链脱保护和从树脂切割 <ol><li>承接测试切割到DISPLA后合成过程中的任何点退房合成（纯度和质量）的进展情况水泥的步骤，加入或去除DDE保护基团的最终序列或之后已经取得进展。 <ol><li>传送从反应容器中约10树脂珠粒成全新的8 15mL的聚丙烯多孔盒。 </li><li>加入1毫升三氟乙酸裂解混合物（含有95％TFA，2.5％的H 2 O，2.5％三异丙基硅烷）和摇动，在室温下90分钟。 </li><li>使用烧结反应容器到10ml圆底烧瓶中过滤从树脂中的TFA裂解混合物。 </li><li>用旋转蒸发器蒸发的裂解混合物和重新溶解得到的油在1毫升乙腈/水以提交LC-MS或HPLC分析。 </li></ol></li><li>为最终裂解:在用于合成的相同多孔聚丙烯反应盒，加入4毫升的TFA裂解混合物（95％TFA，2.5％的H 2 O，2.5％三异丙基硅烷）的并覆盖容器。摇动，在室温下90分钟。 </li><li>第筛选从树脂使用烧结反应容器到50毫升圆底烧瓶中ëTFA裂解混合液。 </li><li>蒸发用旋转蒸发器的裂解混合物。为TFA已被删除后，产品应为油来获得。这个原油有助于TFA除去，加入2毫升无水二乙醚和拟肽应该沉淀。 <ol><li>无论是通过吸移管除去乙醚并丢弃或用旋转蒸发器蒸发。重复乙醚沉淀三次。 </li></ol></li><li>溶解粗拟肽在10ml乙腈/酸化水溶液（50％乙腈，0.1％TFA的水体积/体积）。转移到一个预先称重的容器中，冷冻在20℃冻干，干燥粉末。 </li></ol></li></ol> 2.表征与纯化注:拟肽合成可以监测和使用C18柱和埃尔通过分析型反相HPLC评估最终拟肽ectrospray液体色谱质谱（LC-MS）。对于LC-MS溶剂的所有HPLC溶剂应新鲜配制。 <ol><li> 分析HPLC <ol><li>称取1毫克拟肽的小型玻璃小瓶中。添加乙腈的最小体积溶解并稀释至1毫升的水。确保拟肽完全溶解。 </li><li>注入10微升至分析HPLC（建议梯度0 - 100％溶剂B经30分钟，其中溶剂A = 95％水，5％乙腈，0.05％TFA，溶剂B = 95％乙腈，5％水，0.03％TFA） ，根据制造商的说明。 </li><li>在220nm可视UV光谱。 </li></ol></li><li> ESI LC-MS <ol><li>使1mg / ml的拟肽溶液如在步骤2.1.1。 </li><li>注入1微升到电喷LC-MS，以确定是否在目标拟肽的分子量存在，使用制造商的说明。 </li><li>检查拟肽序列的质量目标使用PEPToid的计算器，按照计算器上的指示。26这个web实用程序还允许在质谱看到任何删除/添加的产品的分配。26 </li></ol></li><li> 制备型反相HPLC <ol><li>溶解粗的拟肽于2ml酸化水/乙腈（95％水，5％的MeCN，0.1％TFA），并使用制造商的方案通过制备型RP-HPLC纯化。确定由分析型HPLC和注入将取决于柱尺寸的量获得的洗提​​时间的梯度。 </li><li>可视化使用检测器设定在220纳米。 </li><li>收集在15ml离心管级分，冷冻于20℃和冷冻干燥。 </li><li>重新分析用LC-MS，并根据制造商的协议分析型HPLC级分。重组纯化分数。 </li></ol></li></ol> 3.抗利什曼原虫寄生墨西哥生物试验驹化:安全评估必须在开始合成前进行利什曼原虫墨西哥被归类于英国和适当的控制措施危险群2病原体必须到位，在开始测试之前。所有工作必须在2级微生物安全柜和佩戴适当的（ 例如，丁腈手套，防护眼镜和白大褂）足够的个人防护装备进行。 <ol><li> 寄生虫的亚文化 <ol><li>通过将小瓶在37℃水浴中进行30秒除霜1毫升-150℃下冷冻的储存墨西哥利什曼原虫 M379。 </li><li>原液与非通气帽转移至10ml施耐德的昆虫培养基（在补充有15％热灭活的胎牛血清和1％青霉素/链霉素pH 7.0）中在25毫升细胞培养物烧瓶中。 </li><li>孵育在26℃下72小时。 </li><li>检查显微镜下（放大400倍）的寄生虫检查的条件。钍安永应该是昆虫阶段前鞭毛体，对数生长期procyclic形式与许多分裂的细胞。 </li><li>维持由子培养寄生虫procyclic前鞭毛体以5×10 5个寄生虫/ ml的每三天的浓度。算使用Neubauer改进血球细胞。 </li></ol></li><li> L.改造墨西哥昆虫阶段到哺乳动物阶段无菌无鞭毛体形式27 <ol><li>在一个25毫升的细胞在Schneider的昆虫介质5×10 5个寄生虫/ ml的准备日志阶段寄生虫的10毫升培养物（在补充有15％热灭活的胎牛血清和1％青霉素/链霉素pH 7.0）中:0天培养瓶用非通风盖。 </li><li>孵育在26℃下48小时。 </li><li>第3天:将10毫升培养到50ml离心管中并离心447 xg离心5分钟。 </li><li>倒掉旧培养基，并加入10 mL施耐德的昆虫媒介（在补充20％H pH值5.5吃灭活的胎牛血清和1％青霉素/链霉素）。轻轻悬浮用吸管在新介质寄生虫沉淀。 </li><li>算使用Neubauer改进血球寄生虫的数目。稀释到pH值为5.5中，并转移到一个25毫升细胞培养瓶5×10 5个寄生虫/ ml的浓度。 </li><li>孵育在26℃下约6天。 </li><li>第9天:检查显微镜（400X）下的寄生虫。它们应该在非复制，感染亚循环前鞭毛体的阶段。 </li><li>算使用Neubauer改进血球寄生虫的数目。稀释到pH值为5.5中5×10 5个寄生虫/ ml的浓度。 </li><li>移除10部ml细胞培养并转移到细胞培养瓶中。孵育在32℃下5天。 </li><li>第14天:检查寄生虫的外观显微镜（400X）下。它们应该在致病无鞭毛体阶段，缺乏鞭毛字符前鞭毛体的信息研究所，并准备检测。 </li></ol></li><li> 在L.细胞毒性测定墨西哥无菌无鞭毛体。 注:类肽的生物测试使用在96孔板中进行高通量测定。这个协议描述L的测试墨西哥无菌无鞭毛，但相同的测定也可以在适当的媒介鞭毛体阶段寄生虫进行。化合物与寄生虫培养的浓度从3 - 100μM的60分钟，然后温育之后10倍稀释24小时。结果通过与基于刃天青细胞存活率溶液（ 例如 ，的AlamarBlue）温育后测量的孔的荧光获得的。 <ol><li>制备化合物储备溶液。称出1毫克使用分析天平最终纯化拟肽产物。分子生物学级二甲基亚砜（DMSO）中的适当体积加至5mM的浓度。使6微升等份冷冻在-20℃。 </li><li>在一式三份的96孔板（推荐板布局中可以看到的结果部分， 图6）制备化合物溶液从100到3微米。加入2微升的各个化合物的5毫米原液在顶行（ 即 A）英寸使用多通道移液器（在pH 5.5和20％牛胎儿血清（FBS），1％青霉素/链霉素（P / S））中添加48微升新鲜施耐德的昆虫媒介顶行。 25微升施耐德的昆虫媒介添加到所有其他行（B - F）。 <ol><li>执行由从顶行分注25微升溶液系列稀释。添加到行下面，混匀。进行稀释，直到最后一行，在过去的25微升溶液应被丢弃。 </li><li>用两性霉素B（5mM的股票）作为阳性对照和DMSO（2％溶液），为一式三份的阴性对照。 </li></ol></li><li>准备寄生虫的解决方案:转移文化50毫升离心管和离心447×g离心5分钟。倒出旧培养基并且加入10 mL施耐德的昆虫培养基（在pH 5.5和20％FBS，1％P / S）。 </li><li>轻轻溶解用吸管在新介质寄生虫的颗粒和使用N​​eubauer改进血球计数。稀释文化，8×10 6寄生虫/毫升。 </li><li>加入25微升L.墨西哥文化到每口井。在32℃孵育板60分钟。 </li><li>从培养箱中取出盘子并从各孔中除去40微升溶液。 </li><li>添加90微升新鲜培养基并在32℃孵育24小时。 </li><li>添加10微升基于刃天青-细胞存活率溶液每个孔中。在32℃孵育4小时。 </li><li>用酶标仪测量荧光（λEX = 540纳米，λEM = 600纳米），按照制造商的说明。通过除去平均背景（从平台仅孔）并相对于到t正常化后的孔的荧光进行比较分析数据他DMSO对照。 </li></ol></li></ol>

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The authors have nothing to disclose.

<html>拟肽正越来越多地应用中的化学生物学和药物化学领域中的研究，例如新的治疗3-5，细胞递送剂18,20和诊断工具。29通常，这些序列是阳离子为病原体以上提供一定程度的选择性的哺乳动物细胞，通过细胞膜穿透并在含水体系有助于溶解的能力。有包含在文献中仅赖氨酸或精氨酸模拟物残基的阳离子类肽的许多例子。然而，到目前为止，同时包含在同一序列中这些阳离子残基的拟肽的合成已经由缺乏合适的合成过程受到阻碍。这里所描述的协议允许以有效的方式来合成混合的阳离子类肽并且是高度期望的，因为它提供了调节两亲性拟肽的生物学和化学特性的路由。 核苷酸"&gt;我们的方法使用一种适应常用submonomer拟肽合成，并允许加入相同序列内两个赖氨酸和精氨酸型单体的，它采用室温联轴器和建立保护基化学所以可以预料，这种方法将成为广大科研组是有用的。要添加精氨酸型残基正交保护，未受保护的二胺标准排水量条件下加入，然后保护与DDE-OH。各种二胺可以是一个60分钟的耦合用于允许从2个碳至6个碳原子长，以进行安装， 即，1,2-二氨基乙烷分别1,6-二氨基己烷。该保护DDE-OH在DMF溶解以及侧链和为非常有效的和有选择性的保护基伯胺。DDE的保护基团离开的仲胺的影响， 例如 ，拟肽链的无保护的N末端30的一个限制是，所有的顺式的论文是手动进行;然而，可以预料，开发的偶合条件使该方法适合于用自动肽/拟肽合成中使用。 DDE的基团的树脂上脱保护是使用在DMF中的2％肼溶液离开，可以在树脂使用的吡唑-1-甲脒将guanidinylated游离胺进行。吡唑-1-甲脒六当量和六当量的DIPEA是按游离胺用在树脂（ 即 ，要安装的六当量为每个n精氨酸型单体）。再次，该反应也是有效的和吡唑-1-甲脒试剂具有在DMF良好的溶解性。反应完成经由LC-MS典型地看到在室温下60分钟后。 由于submonomer方法的通用性，各种各样的伯胺可以在置换工序中使用这样的条件，可能需要进行优化，以提高耦合艾菲效率和整体产物收率或纯度。31对于上面所讨论的序列，没有特别的条件是必要的成功联接。然而，也可使用较长的位移倍或更高的胺浓度为有问题的位移（ 即 ，为不良亲核或空间庞大的胺）。一些胺可以不在DMF完全溶解，在这种情况下，建议以溶解这些在N-甲基-2-吡咯烷酮（NMP），或用于固相反应的其它合适的溶剂，而不是如在submonomer合成先前综合方法的拟肽。32为了将包含在侧链中未受保护的杂原子的单体，使用氯乙酸乙酰已被证明是由其它基团有效的。33此外，其它树脂可以与该方法中使用以产生具有不同的C末端官能拟肽。王树脂和2-氯三苯甲基氯树脂的常规使用submonomer合成拟肽的。例如，该方法已成功地用2-氯三苯甲基氯树脂中的组使用。2不同固体载体将需要不同的加载过程到Rink酰胺这里讨论（取决于所使用的特定树脂），所以这应该与文献进行检查之前的合成。 类似于肽合成，对于拟肽关闭树脂的最终裂解的条件，也可以为特定的序列的优化。在这个协议中，为TFA裂解混合物，使用（用三异丙基硅烷和水作为清除剂）。这里介绍的拟肽仅含有的Boc保护这是一个合理的酸不稳定基团。以确保序列的完全脱保护以保护残基或酸以下不稳定的保护基团的比例更大，更长的切割时间可能是必要的（ 即 ，超过2小时裂解倍推荐用于包含了Pbf或叔府序列TYL酯保护组）。替代清除剂也可用于专门侧链（例如，乙二硫醇或2-巯基乙醇在具有含硫侧链的像半胱氨酸或甲硫氨酸的肽经常使用）。 提出的生物测定是一个标准的细胞毒性试验，可以被改变以适应不同的细胞系。要注意，每个96孔板应包含足够的控制，以允许在所得到的结果的可信度是很重要的。在这种情况下，两性霉素B被用作阳性对照，因为它是用于治疗疾病的已知药物和DMSO用作阴性对照，因为这是用于制造化合物股用于测定的溶剂。如果正在使用其它细胞系，替代方案中，适当的控制，应获得并在使用前进行验证。L.墨西哥是在100和2μm之间的浓度拟肽培养一小时，然后在寄生虫/拟肽溶液是通过稀释十过夜培养的因素（ 即井最初与100μM拟肽股票稀释到10微米）。 细胞活力试剂在测定结束时加入到各孔（总井体积的10％）。可见的颜色变化见过活寄生虫（粉红色），对照孔没有活寄生虫（蓝色）和活寄生虫的中间数之间的频谱井之间。荧光正比于活细胞的数目，并对应于该细胞的代谢活性;刃天青染料（非荧光）通过还原反应在代谢活性细胞转化成荧光试卤灵。34在该试验中，培养时间与细胞活力试剂已为L.优化墨西哥 。与存活率试剂将用于板发生变化的温育时间接种在不同的细胞浓度或者与不同的细胞系（例如，也可使用这种方法与哺乳动物细胞）。取决于所用的确切板读数器，考虑需要采取荧光测量之前进行。在井的任何气泡应去掉，以确保可采取准确的读数。一些板的读者从板的底部，在这种情况下，平底96孔板应使用读取。其他机器可从板的顶部阅读，所以该板的盖子应该测量前被移除。 最后，在将来，这种协议可以用自动化合成仪，其能够在平行使得许多序列兼容。此外，环状拟肽的合成，也可以使用这种方法。该协议应当提供与该可用来访问新颖拟肽支架与两个赖氨酸和精氨酸型单体，其可以是用在许多应用中，包括材料或药物领域的实用合成方法的研究。

类肽（或聚ñ取代的甘氨酸）是一类肽模拟物提供类似性质的肽，因此越来越多地被研究用于医药和材料的应用的。在肽中，每个氨基酸的侧链连接到酰胺主链的α碳;在拟肽侧链被移位到所述主链的氮原子。重要的是，这给拟肽蛋白水解更大的阻力。 拟肽使用由Zuckermann 等人，其中，拟肽单体可以通过附连到一个固体支持物，并使用伯胺的卤素的后续位移胺官能顺序haloacetylation建率先submonomer方法通常合成。1我们小组最近开发一种适应此submonomer方法以允许赖氨酸和精氨酸型拟肽残基被包括为F同一类肽序列内IRST时间。2本手册固相的方法来合成类肽使用市售试剂和通用实验室设备，使之成为广大实验室的访问。拟肽已被证明具有对多种革兰氏阴性菌，革兰氏阳性细菌和真菌种类属于媲美许多已知的抗微生物肽的有希望的活性。3-9 在我们的工作中，拟肽已被用来作为一种新型的抗感染化合物用于治疗被忽视的热带疾病利什曼病，利什曼病5,10是在全球超过80个国家和地方性据估计，超过12万人感染了全球11的病是由由白蛉叮咬传播的原生动物寄生虫引起的。 利什曼原虫属物种可导致皮肤利什曼病，从而导致疤痕和损伤粘膜的条件，或危及生命的内脏花环hmaniasis，这会导致致命的器官损伤。没有疫苗是目前可用于本病与现有的治疗依赖于少数有严重的副作用的药物。此外，对现有药物的耐药性是一个新兴的和严重的问题，所以新的治疗迫切需要有效治疗利什曼病在未来12-16 在这些抗微生物应用中，拟肽通常设计为两亲性与阳离子和疏水单体的混合物。3,4-这可以使拟肽度朝细菌细胞的选择性，减少毒性哺乳动物细胞，并改善它们的分子转运活性17-20中的大多数文献抗感染拟肽的包含被专门由任一氨基官能赖氨酸型单体或精氨酸型残基的阳离子侧链。肽 - 类肽嵌合体，其中所述阳离子链由所述一个的美浓酸赖氨酸或精氨酸，也被合成以审查活性和毒性阳离子基团的效果。21-25 多聚赖氨酸拟肽可以使用市售的Boc-保护的胺容易地合成。聚精氨酸拟肽报告可以利用使用的吡唑-1-甲脒作为胍基剂的方法进行。18然而，这只能在拟肽已从除去树脂和Boc保护的侧链裂解后进行，所以该序列中的每一个赖氨酸型残基被转化为一个精氨酸残基。在努力微调化合物的化学和生物特性，我们开发了一种方法，允许双阳离子官能（ 例如 ，N-赖氨酸和N精氨酸）被包括在首次任何给定的拟肽序列。2 这里，我们描述总重量的合成，纯化和表征Ó包含在同一序列的两个赖氨酸和精氨酸型残基小说类肽。该方法使用与吡唑-1-甲脒作为胍基的试剂上树脂正交Ñ-Boc和N p&#39;-DDE保护。这些拟肽的生物学评价中还针对墨西哥利什曼原虫 ，皮肤利什曼病的病原体的细胞毒性测定法描述。这提供了具有双阳离子官能访问拟肽，并评估其生物活性的实用方法。据预计，该方法将由拟肽社区在未来有助于两亲性拟肽的合成。

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			<td height="63" style="height:63px;width:216px;">Polypropylene solid phase extraction cartridges with two frits</td>
			<td style="width:144px;">Crawford Scientific</td>
			<td style="width:119px;">12131017 and 12131015</td>
			<td style="width:1791px;">20 mL and 6 mL cartridges used in this preparation</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Trifluoroacetic acid</td>
			<td style="width:144px;">Tokyo Chemical Industry (Europe)</td>
			<td style="width:119px;">T0431-100g</td>
			<td>&#62;98%; CAUTION can cause severe burns and respiratory irritation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">N-N&#39;-diisopropylcarbodiimide</td>
			<td style="width:144px;">Sigma Aldrich</td>
			<td>38370-100ML</td>
			<td>&#62;98%; CAUTION hazardous to eyes, skin and via respiratory inhalation, may also cause sensitisation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Dimethylformamide</td>
			<td style="width:144px;">Fischer Scientific</td>
			<td align="right" style="width:119px;">10346180</td>
			<td>HPLC grade; CAUTION suspected teratogen</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Acetonitrile</td>
			<td style="width:144px;">Fischer Scientific</td>
			<td align="right" style="width:119px;">10407440</td>
			<td>HPLC grade</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Dichloromethane</td>
			<td style="width:144px;">Fischer Scientific</td>
			<td align="right" style="width:119px;">10354263</td>
			<td>99.8%; CAUTION suspected carcinogen</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Bromacetic acid</td>
			<td style="width:144px;">Sigma Aldrich</td>
			<td>17000-100G</td>
			<td>&#62;99%; ; CAUTION causes burns and hazardous to skin, eyes and respiratory tract</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">(S)-(-)-alpha methylbenzylamine</td>
			<td style="width:144px;">Sigma Aldrich</td>
			<td>115568-100G</td>
			<td>98%; CAUTION harmful if swallowed, toxic in contact with skin, causes severe skin burns and eye damage, used to synthesise the Nspe monomer</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">N-Boc 1,4 diaminobutane</td>
			<td style="width:144px;">Tokyo Chemical Industry (Europe)</td>
			<td style="width:119px;">A1373-25g</td>
			<td>&#62;98%; CAUTION causes severe skin burns and eye damage, used to synthesise the NLys monomer</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">N-Boc 1,2 diaminoethane</td>
			<td style="width:144px;">Tokyo Chemical Industry (Europe)</td>
			<td>A1371-25g</td>
			<td>&#62;97%; CAUTION causes severe skin burns and eye damage, used to synthesise the Nae monomer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">1,2 diaminobutane</td>
			<td style="width:144px;">Sigma Aldrich</td>
			<td>D13208-100G</td>
			<td>99%; CAUTION flammable liquid and vapour, harmful if swallowed or inhaled, toxic in contact with skin, causes severe skin burns and eye damage, used in the synthesis of the NhArg monomer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">1,4 diaminoethane</td>
			<td style="width:144px;">Sigma Aldrich</td>
			<td>03550-250ML</td>
			<td>&#62;99.5%; CAUTION flammable liquid and vapour, harmful if swallowed or inhaled, toxic in contact with skin, causes severe skin burns and eye damage, used in the synthesis of the NnArg monomer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">1H-pyrazole-1-carboxamidine HCl</td>
			<td style="width:144px;">Sigma Aldrich</td>
			<td>402516-10G</td>
			<td>as HCl salt; CAUTION harmful if swallowed and may cause an allergic skin reaction, causes serious eye damage</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Hydrazine monohydrate</td>
			<td style="width:144px;">Sigma Aldrich</td>
			<td>207942-5G</td>
			<td>reagent grade; CAUTION suspected carcinogen, fatal if inhaled and causes severe burns to skin and eyes</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">2-acetyl dimedone</td>
			<td style="width:144px;">Novabiochem</td>
			<td align="right">8510150005</td>
			<td>Dde-OH</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Rink Amide resin</td>
			<td style="width:144px;">Novabiochem</td>
			<td align="right">8551190005</td>
			<td>100-200 mesh, high loading&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Piperidine</td>
			<td style="width:144px;">Sigma Aldrich</td>
			<td>411027-1L</td>
			<td>&#62;99.5%, a controlled substance, so adequate permission must be obtained before purchase; CAUTION highly flammable liquid and vapour, harmful if swallowed and toxic in contact with skin or if inhaled, causes severe skin burns and eye damage, harmful to aquatic life with long lasting effects</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Triisopropylsilane</td>
			<td style="width:144px;">Sigma Aldrich</td>
			<td>233781-50G</td>
			<td>98%; CAUTION flammable liquid and vapour, causes skin irritation and serious eye irritation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">alamarBlue</td>
			<td style="width:144px;">ThermoFischer&#160;</td>
			<td style="width:119px;">DAL1025</td>
			<td>Not classified as hazardous</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Schneider&#39;s Insect Medium</td>
			<td style="width:144px;">Sigma Aldrich</td>
			<td>S9895-1L</td>
			<td>Powdered, medium must be made prior to use following manafacturers instructions; allow to warm to room temperature before use in biological assays</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">96 well plates</td>
			<td style="width:144px;">VWR</td>
			<td style="width:119px;">734-1793</td>
			<td>Flat bottom (to allow fluorescence measurement from the bottom), tissue-culture treated</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Solvent reservoirs</td>
			<td style="width:144px;">VWR</td>
			<td>613-1182</td>
			<td>Used with multi channel pipette</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Multi channel pipette</td>
			<td style="width:144px;">Eppendorf</td>
			<td align="right" style="width:119px;">3122000043</td>
			<td style="width:1791px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Pipette tips</td>
			<td style="width:144px;">Starlab Group</td>
			<td>S1111-3810, S1113-1810, S1111-6810</td>
			<td>Volume of tip dependent on pipette used. 10 &#181;L, 10 - 200 &#181;L and 1000 &#181;L recommended for assays</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">25 cm3 cell culture flasks</td>
			<td style="width:144px;">VWR</td>
			<td style="width:119px;">734-2312</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">50 mL centrifuge tubes</td>
			<td style="width:144px;">VWR</td>
			<td style="width:119px;">525-0791</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:216px;">dimethylsulphoxide (molecular biology grade)</td>
			<td style="width:144px;">Sigma Aldrich</td>
			<td>D8418-50ML</td>
			<td>Not classified as hazardous</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Heat Inactivated Fetal Bovine Serum</td>
			<td style="width:144px;">ThermoFischer&#160;</td>
			<td style="width:119px;">10082139-100mL</td>
			<td>Gibco</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Penicillin/Streptomycin</td>
			<td style="width:144px;">ThermoFischer&#160;</td>
			<td style="width:119px;">15140148-20mL</td>
			<td style="width:1791px;">Abbreviation: P/S</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Amphotericin B</td>
			<td style="width:144px;">Sigma Aldrich</td>
			<td>46006-100mg</td>
			<td>Amphotericin B trihydrate, VetranalTM analytical standard</td>
		</tr>
	</tbody>
</table>

an efficient method for the synthesis of peptoids with mixed lysine-type/arginine-type monomers and evaluation of their anti-leishmanial activity

This protocol describes the manual solid-phase synthesis of linear peptoids that contain two differently functionalized cationic monomers. In this procedure amino functionalized 'lysine' and guanido functionalized 'arginine' peptoid monomers can be included within the same peptoid sequence. This procedure uses on-resin (N-(1-(4,4-dimethyl-2,6-dioxocyclohexylidene)ethyl) or Dde protection, orthogonal conditions to the Boc protection of lysine monomers. Subsequent deprotection allows an efficient on-resin guanidinylation reaction to form the arginine residues. The procedure is compatible with the commonly used submonomer method of peptoid synthesis, allowing simple peptoids to be made using common laboratory equipment and commercially available reagents. The representative synthesis, purification and characterization of two mixed peptoids is described. The evaluation of these compounds as potential anti-infectives in screening assays against Leishmania mexicana is also described. The protozoan parasite L. mexicana is a causative agent of cutaneous leishmaniasis, a neglected tropical disease that affects up to 12 million people worldwide.

作为一个代表性的结果，含有两个赖氨酸型单体和双精氨酸型单体的每个将描述两个12残基类肽的合成和表征。从细胞毒性测定随后的结果也示。 两个拟肽[（N nArgñSPEñSPE）（N AEñSPEñSPE）] 2（a）和[（N HARGñSPEñSPE）（N赖氨酸ñSPEñSPE） 图2（b）的合成使用120毫克的Rink酰胺树脂每一个（负荷= 0.79毫摩尔/克）。所有乙酰化和位移步骤，如上所述，与可商购的所有试剂进行。对这些残基，下面的胺在置换工序中使用:N SPE（S） - （ - ） - α甲基苄胺，N AE -N - （ 叔丁氧羰基）-1,2-直径inoethane，N 赖氨酸 N - （ 叔丁氧羰基）-1,4-二氨基丁烷。为精氨酸衍生物残基，下面的未保护的二胺偶合:N HARG 1,4-二氨基丁烷或N nArg 1,2-二氨基乙烷，接着在树脂保护用2- acetyldimedone（DDE-OH）。整个序列已被合成后，DDE的肼脱保护产生游离胺guanidinylate。 <img alt="图1" src="/files/ftp_upload/54750/54750fig1.jpg" /> 图1.拟肽结构（一）[（N nArgñSPEñSPE）（N AEñSPEñSPE）] 2和（b）[（N HARGñSPEñSPE）（N <stro<html> NG&gt;赖氨酸ñSPEñSPE）] 2。 <a href="https://www.jove.com/files/ftp_upload/54750/54750fig1large.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/54750/54750fig2.jpg" /> 图2.用于合成混合精氨酸/赖氨酸拟肽。我的方法 。标准排水量一步与二胺submonomer方法; II。 DDE的-OH，90分钟除了保护游离胺;三。进一步的添加使用submonomer方法来扩展类肽链; IV。使用DDE在DMF 2％肼脱保护;诉与在DMF吡唑-1-甲脒和DIPEA树脂游离胺的胍基;六。由Boc-基团的树脂和脱保护的酸性裂解。large.jpg"目标="_空白"&gt;点击此处查看该图的放大版本。 （一）154毫克，（二）163毫克:以下从树脂并冻干裂解，将粗产物，为白色粉末获得。与使用最多50毫克注射一个LC泵与一个分析柱的UV-Vis检测器（λ= 250纳米），250毫米×10毫米，5微米所述产物通过RP-HPLC纯化;流速= 2毫升/分。对应于目标质量级分合并并为白色粉末获得:（1）54毫克（二）65毫克，对分数&gt; 90％纯度约30％的最终收率。 纯化后的最终化合物的身份通过LC-MS（ 见图3）使用配备UPLC和光电二极管阵列检测器的三重四极质谱仪证实。准确的质谱用在T相同的光谱仪进行<html>他[M + 2H] 2+。以下计算并密切协议（ 图4）被发现观察质量:（1）计算值= 896.0026 amu的，实测值= 896.0038 amu的; （二）计算值= 952.0691阿拉伯马格里布联盟，实测值= 952.0730 AMU。 <img alt="图3" src="/files/ftp_upload/54750/54750fig3.jpg" /> 图3. LC-MS用于纯化的拟肽（a）中 M / Z = 1,792和（b）M / Z = 1903。其中，最上面是TIC，中间LC-MS谱，在220nm紫外线下色谱图。 <a href="https://www.jove.com/files/ftp_upload/54750/54750fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图4" src="/files/ftp_upload/54750/54750fig4.jpg" /> 图对拟肽4.精确质谱数据（a）和（b）中。"https://www.jove.com/files/ftp_upload/54750/54750fig4large.jpg"目标="_空白"&gt;点击此处查看该图的放大版本。 产品纯度，采用（在分析柱和紫外可见检测器LC泵，4.6毫米×100毫米，3.5微米;流速= 1毫升/分钟）的分析型RP-HPLC评估，并在220nm，吸光度可视酰胺主链。 图5a和5b示出该化合物是均匀的。 <img alt="图5" src="/files/ftp_upload/54750/54750fig5.jpg" /> 图5.分析性HPLC为纯化的拟肽（a）和（b）的有在30分钟内，柱烘箱的梯度0-100％B，在40℃（A = 95％H 2 O，5％的MeCN，0.05 ％TFA; B = 95％MeCN中，5％的H 2 O，0.03％TFA） <a href="https://www.jove.com/files/ftp_upload/54750/54750fig5large.jpg" target="_blank">请CL</a>ICK这里查看该图的放大版本。 纯化的拟肽（a）和（b）如针对L。细胞毒性试验进行了测试墨西哥无菌无鞭毛体。 L.冰冻股票墨西哥解冻并转化为无鞭毛体阶段准备用于测定。除霜后72小时，这些寄生虫应在其procyclic形式昆虫阶段前鞭毛体，在对数期与许多分裂的细胞。在这个阶段，寄生虫可以使用上述27处的改造9日的pH和温度偏移被转化成无鞭毛体阶段，寄生虫将在非复制传染性亚循环前鞭毛体的阶段。最后，在第14天，这些寄生虫应在寄生虫缺乏前鞭毛体的特性鞭毛致病无鞭毛体的阶段。28 5化合物的10mM储备溶液在细胞培养级DMSO中制成，并在一式三份测试了最少两次以确保可靠的数据集收集。代表性的96孔板计划示出在图6中。在该试验结束时，细胞存活率试剂加入到每个孔中，所述荧光测定如所述计算寄生虫在测试的各浓度的生存能力（ 见图7 ）。 ED 50值计算成拟肽（一）&gt; 100μM和拟肽（B）分别为37微米。误差条绘制，显示为标准偏差的井之间的变化，并可以看出，这些都是合理的最酒吧。 <img alt="图6" src="/files/ftp_upload/54750/54750fig6.jpg" /> 图6.代表96孔板计划2拟肽溶液（包括阳性和阴性对照）一个细胞毒性测定。配有+ =中等（Schneider的昆虫培养基，pH值5.5，20％FBS，1％P / S）。L. MEX。 =8×10 6 / ml的寄生虫文化MED +。 AmphoB = 5mM的DMSO中。拟肽= 5mM储备溶液在DMSO中。井空应该包含无菌水。 <a href="https://www.jove.com/files/ftp_upload/54750/54750fig6large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图7" src="/files/ftp_upload/54750/54750fig7.jpg" /> 图7.对L.细胞毒性试验结果墨西哥无菌使用拟肽无鞭毛体寄生虫（a）和（b）中。由此可以看出，类肽（b）是（a）于还原活寄生虫的百分比，与具有剂量依赖性效应的两种化合物比拟肽更加有效。绘制误差条显示为一个标准差井之间的变化。 <a href="https://www.jove.com/files/ftp_upload/54750/54750fig7large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about An Efficient Method for the Synthesis of Peptoids with Mixed Lysine-type/Arginine-type Monomers and Evaluation of Their Anti-leishmanial Activity at JoVE.com

An Efficient Method for the Synthesis of Peptoids with Mixed Lysine-type/Arginine-type Monomers and Evaluation of Their Anti-leishmanial Activity

A protocol to synthesize peptoids with mixed cationic functionality in the same sequence is presented (lysine- and arginine-type monomers). Subsequent testing of these compounds against Leishmania mexicana, the protozoan parasites that cause cutaneous leishmaniasis, is also described.

对于拟肽与他们抗利什曼原虫活性混合赖氨酸型/精氨酸型单体及评价合成的有效方法

This protocol describes the manual solid-phase synthesis of linear peptoids that contain two differently functionalized cationic monomers. In this ...

an-efficient-method-for-synthesis-peptoids-with-mixed-lysine

Research

JoVE Journal

Biochemistry

12.0K 次观看.  Durham University. A protocol to synthesize peptoids with mixed cationic functionality in the same sequence is presented (lysine- and arginine-type monomers). Subsequent testing of these compounds against Leishmania mexicana, the protozoan parasites that cause cutaneous leishmaniasis, is also described.

视频: An Efficient Method for the Synthesis of Peptoids with Mixed Lysine-type/Arginine-type Monomers and Evaluation of Their Anti-leishmanial Activity

Preparation and In Vivo Use of an Activity-based Probe for N-acylethanolamine Acid Amidase

Activity-based protein profiling (ABPP) is a method for the identification of an enzyme of interest in a complex proteome through the use of a chemical probe that targets the enzyme&#39;s active sites. A reporter tag introduced into the probe allows for the detection of the labeled enzyme by in-gel fluorescence scanning, protein blot, fluorescence microscopy, or liquid chromatography-mass spectrometry. Here, we describe the preparation and use of the compound ARN14686, a click chemistry activity-based probe (CC-ABP) that selectively recognizes the enzyme N-acylethanolamine acid amidase (NAAA). NAAA is a cysteine hydrolase that promotes inflammation by deactivating endogenous peroxisome proliferator-activated receptor (PPAR)-alpha agonists such as palmitoylethanolamide (PEA) and oleoylethanolamide (OEA). NAAA is synthesized as an inactive full-length proenzyme, which is activated by autoproteolysis in the acidic pH of the lysosome. Localization studies have shown that NAAA is predominantly expressed in macrophages and other monocyte-derived cells, as well as in B-lymphocytes. We provide examples of how ARN14686 can be used to detect and quantify active NAAA ex vivo in rodent tissues by protein blot and fluorescence microscopy.

Preparation and In Vivo Use of an Activity-based Probe for N-acylethanolamine Acid Amidase

Here, we describe the preparation and use of an activity-based probe (ARN14686, undec-10-ynyl-N-[(3S)-2-oxoazetidin-3-yl]carbamate) that allows for the detection and quantification of the active form of the proinflammatory enzyme N-acylethanolamine acid amidase (NAAA), both in vitro and ex vivo.

制备及<em&gt;在体内</em&gt;使用基于活动的探针对<em&gt;ñ</em&gt; -acylethanolamine酸酰胺酶

Activity-based protein profiling (ABPP) is a method for the identification of an enzyme of interest in a complex proteome through the use of a chemical ...

科研

生物化学

A Facile and Efficient Approach for the Production of Reversible Disulfide Cross-linked Micelles

Nanomedicine is an emerging form of therapy that harnesses the unique properties of particles that are nanometers in scale for biomedical application. Improving drug delivery to maximize therapeutic outcomes and to reduce drug-associated side effects are some of the cornerstones of present-day nanomedicine. Nanoparticles in particular have found a wide application in cancer treatment. Nanoparticles that offer a high degree of flexibility in design, application, and production based on the tumor microenvironment are projected to be more effective with rapid translation into clinical practice. The polymeric micellar nano-carrier is a popular choice for drug delivery applications.
In this article, we describe a simple and effective protocol for synthesizing drug-loaded, disulfide cross-linked micelles based on the self-assembly of a well-defined amphiphilic linear-dendritic copolymer (telodendrimer, TD). TD is composed of polyethylene glycol (PEG) as the hydrophilic segment and a thiolated cholic acid cluster as the core-forming hydrophobic moiety attached stepwise to an amine-terminated PEG using solution-based peptide chemistry. Chemotherapy drugs, such as paclitaxel (PTX), can be loaded using a standard solvent evaporation method. The O2-mediated oxidation was previously utilized to form intra-micellar disulfide cross-links from free thiol groups on the TDs. However, the reaction was slow and not feasible for large-scale production. Recently, an H2O2-mediated oxidation method was explored as a more feasible and efficient approach, and it was 96 times faster than the previously reported method. Using this approach, 50 g of PTX-loaded, disulfide cross-linked nanoparticles have been successfully produced with narrow particle size distribution and high drug loading efficiency. The stability of the resulting micelle solution is analyzed using disrupting conditions such as co-incubation with a detergent, sodium dodecyl sulfate, with or without a reducing agent. The drug-loaded, disulfide cross-linked micelles demonstrated less hemolytic activity when compared to their non-cross-linked counterparts.

To deliver cancer drugs to tumor sites with high specificity and reduced side effects, new methods based on nanoparticles are required. Here, we describe disulfide cross-linked micelles that can be easily prepared by hydrogen peroxide-mediated oxidation and are able to dissociate efficiently under a reducing tumor environment to release payloads.

可逆二硫键交联胶束生产的简便和有效途径

Nanomedicine is an emerging form of therapy that harnesses the unique properties of particles that are nanometers in scale for biomedical application. ...

An Efficient Method for the Isolation of Highly Purified RNA from Seeds for Use in Quantitative Transcriptome Analysis

Plant seeds accumulate large amounts of storage reserves comprising biodegradable organic matter. Humans rely on seed storage reserves for food and as industrial materials. Gene expression profiles are powerful tools for investigating metabolic regulation in plant cells. Therefore, detailed, accurate gene expression profiles during seed development are required for crop breeding. Acquiring highly purified RNA is essential for producing these profiles. Efficient methods are needed to isolate highly purified RNA from seeds. Here, we describe a method for isolating RNA from seeds containing large amounts of oils, proteins, and polyphenols, which have inhibitory effects on high-purity RNA isolation. Our method enables highly purified RNA to be obtained from seeds without the use of phenol, chloroform, or additional processes for RNA purification. This method is applicable to Arabidopsis, rapeseed, and soybean seeds. Our method will be useful for monitoring the expression patterns of low level transcripts in developing and mature seeds.

We have succeeded in establishing a method for RNA isolation from plant seeds containing large amounts of oils, proteins, and polyphenols, which have inhibitory effects on high-purity RNA isolation. Our method is suitable for monitoring the expression of genes with low level transcripts in seeds.

对于高度纯化RNA的种子定量转录组分析的隔离中使用的有效方法

Plant seeds accumulate large amounts of storage reserves comprising biodegradable organic matter. Humans rely on seed storage reserves for food and as ...

Synthesis of 1,2-Azaborines and the Preparation of Their Protein Complexes with T4 Lysozyme Mutants

We describe a general synthesis of 1,2-azaborines using standard air-free techniques and protein complex preparation with T4 lysozyme mutants by vapor diffusion. Oxygen- and moisture-sensitive compounds are prepared and isolated under an inert atmosphere (N2) using either a vacuum gas manifold or a glove box. As an example of azaborine synthesis, we demonstrate the synthesis and purification of the volatile N-H-B-ethyl-1,2-azaborine by a five-step sequence involving distillation and column chromatography for the isolation of products. T4 lysozyme mutants L99A and L99A/M102Q are expressed with Escherichia coli RR1 strain. Standard protocols for chemical cell lysis followed by purification using carboxymethyl ion exchange column affords protein of sufficiently high purity for crystallization. Protein crystallization is performed in various concentrations of precipitant at different pH ranges using the hanging drop vapor diffusion method. Complex preparation with the small molecules is carried out by vapor diffusion method under an inert atmosphere. X-ray diffraction analysis of the crystal complex provides unambiguous structural evidence of binding interactions between the protein binding site and 1,2-azaborines.

A protocol for the synthesis of 1,2-azaborines and the preparation of their protein complexes with T4 lysozyme mutants is presented.

1,2- Azaborines的合成及其蛋白复合物的用T4溶菌酶突变体的制备

We describe a general synthesis of 1,2-azaborines using standard air-free techniques and protein complex preparation with T4 lysozyme mutants by vapor ...

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 ...

Method for Efficient Refolding and Purification of Chemoreceptor Ligand Binding Domain

Identification of natural ligands of chemoreceptors and structural studies aimed at elucidation of the molecular basis of the ligand specificity can be greatly facilitated by the production of milligram amounts of pure, folded ligand binding domains. Attempts to heterologously express periplasmic ligand binding domains of bacterial chemoreceptors in Escherichia coli (E. coli) often result in their targeting into inclusion bodies. Here, a method is presented for protein recovery from inclusion bodies, its refolding and purification, using the periplasmic dCACHE ligand binding domain of Campylobacter jejuni (C. jejuni) chemoreceptor Tlp3 as an example. The approach involves expression of the protein of interest with a cleavable His6-tag, isolation and urea-mediated solubilisation of inclusion bodies, protein refolding by urea depletion, and purification by means of affinity chromatography, followed by tag removal and size-exclusion chromatography. The circular dichroism spectroscopy is used to confirm the folded state of the pure protein. It has been demonstrated that this protocol is generally useful for production of milligram amounts of dCACHE periplasmic ligand binding domains of other bacterial chemoreceptors in a soluble and crystallisable form.

A procedure is presented for the refolding of the dCACHE periplasmic ligand binding domain of Campylobacter jejuni chemoreceptor Tlp3 from inclusion bodies and the purification to yield milligram quantities of protein.

化学感受器配体结合域的高效复用与纯化方法

Identification of natural ligands of chemoreceptors and structural studies aimed at elucidation of the molecular basis of the ligand specificity can be ...

Enrichment of Native Lipoprotein Particles with microRNA and Subsequent Determination of Their Absolute/Relative microRNA Content and Their Cellular Transfer Rate

Lipoprotein particles are predominately transporters of lipids and cholesterol in the bloodstream. Furthermore, they contain small amounts of strands of noncoding microRNA (miRNA). In general, miRNA alters the protein expression profile due to interactions with messenger-RNA (mRNA). Thus, knowledge of the relative and absolute miRNA content of lipoprotein particles is essential to estimate the biological effect of cellular particle uptake. Here, a quantitative real-time polymerase chain reaction (qPCR)-based protocol is presented to determine the absolute miRNA content of lipoprotein particles&#8212;exemplified shown for native and miRNA-enriched lipoprotein particles. The relative miRNA content is quantified using multiwell microfluidic array cards. Furthermore, this protocol allows scientists to estimate the cellular miRNA and, thus, the lipoprotein particle uptake rate. A significant increase of the cellular miRNA level is observable when using high-density lipoprotein (HDL) particles artificially loaded with miRNA, whereas incubation with native HDL particles yields no significant effect due to their rather low miRNA content. In contrast, the cellular uptake of low-density lipoprotein (LDL) particles&#8212;neither with native miRNA nor artificially loaded with it&#8212;did not alter the cellular miRNA level.

Here, a quantitative real-time polymerase chain reaction-based protocol is presented for the determination of the native micro-RNA content (absolute/relative) of lipoprotein particles. In addition, a method for increasing the micro-RNA level, as well as a method for determining the cellular uptake rate of lipoprotein particles, is demonstrated.

微 Rna 对原生脂蛋白颗粒的富集及其与绝对相对微 Rna 含量及细胞转移速率的测定

Lipoprotein particles are predominately transporters of lipids and cholesterol in the bloodstream. Furthermore, they contain small amounts of strands of ...

Deciphering Molecular Mechanism of Histone Assembly by DNA Curtain Technique

Chromatin is a higher-order structure that packages eukaryotic DNA. Chromatin undergoes dynamic alterations according to the cell cycle phase and in response to environmental stimuli. These changes are essential for genomic integrity, epigenetic regulation, and DNA metabolic reactions such as replication, transcription, and repair. Chromatin assembly is crucial for chromatin dynamics and is catalyzed by histone chaperones. Despite extensive studies, the mechanisms by which histone chaperones enable chromatin assembly remains elusive. Moreover, the global features of nucleosomes organized by histone chaperones are poorly understood. To address these problems, this work describes a unique single-molecule imaging technique named DNA curtain, which facilitates the investigation of the molecular details of nucleosome assembly by histone chaperones. DNA curtain is a hybrid technique that combines lipid fluidity, microfluidics, and total internal reflection fluorescence microscopy (TIRFM) to provide a universal platform for real-time imaging of diverse protein-DNA interactions.Using DNA curtain, the histone chaperone function of Abo1, the Schizosaccharomyces pombe bromodomain-containing AAA+ ATPase, is investigated, and the molecular mechanism underlying histone assembly of Abo1 is revealed. DNA curtain provides a unique approach for studying chromatin dynamics.

DNA curtain, a high-throughput single-molecule imaging technique, provides a platform for real-time visualization of diverse protein-DNA interactions. The present protocol utilizes the DNA curtain technique to investigate the biological role and molecular mechanism of Abo1, a Schizosaccharomyces pombe bromodomain-containing AAA+ ATPase.

DNA帘法破译组蛋白组装的分子机制

Chromatin is a higher-order structure that packages eukaryotic DNA. Chromatin undergoes dynamic alterations according to the cell cycle phase and in ...

A Nanobar-Supported Lipid Bilayer System for the Study of Membrane Curvature Sensing Proteins in vitro

Membrane curvature plays important roles&#160;in various essential processes of cells, such as cell migration, cell division, and vesicle trafficking. It is not only passively generated by cellular activities, but also actively regulates protein interactions and is involved in many intracellular signaling. Thus, it is of great value to examine the role of membrane curvature in regulating the distribution and dynamics of proteins and lipids. Recently, many techniques have been developed to study the relationship between the curved membrane and protein in vitro. Compared to traditional techniques, the newly developed nanobar-supported lipid bilayer (SLB) offers both high-throughput and better accuracy in membrane curvature generation by forming a continuous lipid bilayer on patterned arrays of nanobars with a pre-defined membrane curvature and local flat control. Both the lipid fluidity and protein sensitivity to curved membranes can be quantitatively characterized using fluorescence microscopy imaging. Here, a detailed procedure on how to form a SLB on fabricated glass surfaces containing nanobar arrays&#160;and the characterization of curvature-sensitive proteins on such SLB are introduced. In addition, protocols for nanochip reusing and image processing are covered. Beyond the nanobar-SLB, this protocol is readily applicable to all types of nanostructured glass chips for curvature sensing studies.

A Nanobar-Supported Lipid Bilayer System for the Study of Membrane Curvature Sensing Proteins in vitro

Here, a nanobar-supported lipid bilayer system is developed to provide a synthetic membrane with a defined curvature that enables the characterization of proteins with curvature sensing ability in vitro.

纳米巴支持的脂质双层系统，用于体外研究膜曲率传感蛋白

Membrane curvature plays important roles&#160;in various essential processes of cells, such as cell migration, cell division, and vesicle trafficking. It ...