笔者想感谢的持续财政支持（译文-0742365）和国家科学基金会在Bruker APEX II衍射仪（译文-0443233），并在Bruker Microtof-II质谱仪（译文-0741793）购买。笔者也想感谢他们使用的微团的Quattro-LC质谱仪欧夏令时设施。

1。准备铌管<ol><li>切割铌（Nb）管前，准备NB清洗解决方案。在500毫升的塑料瓶，测出并添加通过测量缸以下的股票解决方案，收到100毫升：110毫升以H 2 SO 4，其次是硝 ​​酸 50毫升，40毫升高频。拌匀，并允许使用前达到室温。 </li><li>测量铌管，长4.5厘米，切管刀。避免油管扭结。重复3次以上。 </li><li>在通风良好的油烟罩，放置四个铌管，长度在100毫升的塑料烧杯。铌清洗液倒入烧杯，直到完全覆盖管。关闭油烟罩的窗扇。等待12至20秒，或直到棕色的氧化亚氮气体被释放。立即装满水的烧杯中，并允许达到室温。 </li><li>使用塑料皮带删除铌管，洗几次水，然后丙酮，在高温烘箱干燥。 （仔细与氢氧化钾中和后处理的溶液）。 </li><li>以铌管，烤箱，让冷却到室温。用老虎钳的交手卷曲各管的一端约1厘米。略微弯曲的边缘，约1毫米。 </li><li>开启真空泵电弧焊机和氩气瓶。夹紧四个铌管，在交错的位置，在电弧焊机的持有人。中心在电弧焊机的铜块（散热片），并慢慢调整铌管顶部。关闭和取缔关闭的门电弧焊机。 </li><li>调整焊接小费铌管的高度。真空阀门关闭（确保氩气填充阀是接近）。允许真空度达到低于100毫米汞柱（理想：30毫米汞柱;等待时间：30 - 40分钟），以完全撤离所有空气和水分室和铌管。 </li><li>当达到30 - 60毫米汞柱的真空，关闭真空阀门，慢慢打开阀门，氩气瓶储值无线氩日至7英寸汞柱。关闭氩气阀门。现在应该下局部真空系统。 </li><li>把认证的橡胶焊接手套。位置保护焊电弧焊机的窗口变黑了玻璃。打开设置源在15（厚的铌管使用20 - 55 A号）。由于焊接的尖端接触铌管，焊接电弧形式，提高管超过1厘米，慢慢地扫上面的管铌完全融化和密封。重复剩余铌管。关闭电源，让系统达到室温（15 - 30分钟）。 </li><li>取出钳弧焊机门。慢慢打开氩气阀门和氩气填充，直到门被打开。 </li><li>取出电雕刻铌管和标签A，B，C和D，分别。放回干燥箱，直到在前厅的drybox 30下抽真空，热干燥和地点 - 45分钟。 </li><li>高纯度氮气/氩气笔芯的小接待室。到drybo带来四个标记的铌管X。现在已经准备好要加载的容器。 </li></ol> 2。载入中铌管：准备钾K 4个9 <ol><li>皮的平衡，然后测量156，镁，钾（4毫摩尔）。用锅铲，仔细铌管插入它推到了底部。 </li><li>重达653毫克葛（9毫摩尔）和顶部钾金属管插入。 </li><li>仔细卷曲的NB用虎钳夹具管的开口端和略微弯曲的边缘。重复其他三个铌管。将惰性气体] [下所有四个管在一个罐子里，关闭罐子放置在框中前厅，以带出。 </li><li>步骤1.5 - 1.8，焊缝边缘的四个铌管。现在已经准备好被密封在石英管装管。 </li></ol> 3。准备通过熔融石英玻璃管吹<ol><li>使用圆形玻璃看到削减10-14“大石英管件（内径/外径= 20/22毫米）和介质的石英管（7/9毫米）。切也结束反渗透与球关节。与水和丙酮彻底清洗。在玻璃干燥炉，直到完全干燥的地方。烘箱取出，并允许达到室温。 </li><li>氢/氧气的火炬点燃一名前锋，并慢慢增加氧气流量变热的蓝色火焰。在漆黑的安全眼镜，把前插入任何石英管火焰。 </li><li>在火焰中插入大型管，一端慢慢地转动，让管收缩到一个瓶颈，然后完全关闭。使用备用介质的石英管，作为塑造杆拉的“白热化”（通过安全眼镜）石英管的一端关闭的开放，更迅速。 </li><li>封开后，附上打击软管橡胶隔在开盖吹管插入口（不吹呢）。而旋转的火焰密封端，稍微吹保持正压，防止倒塌的玻璃。接下来，一个小洞是准备在中心。 </li><li>调整气体流量来创造吃了尖锐的蓝色火焰在封闭端中心和重点火焰。当有针对性的现场变得白热化，吹努力创造一个大的泡沫或开口。打破在实验室工作台上轻轻刮泡沫。插入到火焰和0.7开洞 - 1厘米而燃烧石墨铰边缘。这部分（身体）是现在已经准备好要附加到的小管（颈）。 </li><li>关闭一端橡胶隔膜的脖子上。身体（口喉舌，另一只手拿着准备炸毁），另一方面颈部慢慢两端同时旋转的火焰。将两端和整顿外的火焰。 </li><li>调整火焰大小，以尖锐的，慢慢地工作，每个季度密封联合，以确保无孔和气泡。保持正压，以防止倒塌的玻璃。取下脖子上的隔膜。现在已经准备好要附加到球联合颈部。 </li><li>使用隔，靠近球的联合开幕式。附加关节球头颈部following步骤3.5 - 3.6。 </li><li>插入四个铌管打开身体年底。球接头重视打击软管年底。慢慢靠近身体末端打开下面的步骤3.3。关闭的火焰，并关闭所有气瓶。允许石英/石英外套降温。 </li><li>倒入稀释铌清洗液（2·H 2 0：1解决方案，是指为1.1）的硅胶管，并保持3 - 5分钟，直到铌管有光泽，无任何氧化的领域。用蒸馏水和丙酮冲洗3次，每次。允许完全干燥。铌管现在在石英/石英外套，准备进行疏散，并封锁了。 </li></ol> 4。密封的熔融石英管采用高真空线<ol><li>打开泵真空线。充满液氮的杜瓦瓶。打开水银扩散泵和冷却水。允许至少30分钟的回流。 </li><li>石英外套大衣球关节与高真空硅GREASE。附加到真空线。 30分钟完全撤离管。 </li><li>使用特斯拉线圈，检查有无渗漏/孔。 </li><li>放在硼的安全眼镜。打开小温室气体/氧气火炬。慢慢从管底颈部传递圣火，确保所有空气和水分了。 </li><li>慢慢越过铌管的火焰，轻轻加热1 - 2分钟。小心，不要让玻璃白热化，因为它会崩溃，因为它是高真空拉。关闭火炬，让石英外套，以达到室温（15 - 30分钟）。 </li><li>重复步骤4.4。加大到一个尖锐的热蓝色的火焰。慢慢封锁低于球管和颈联合。 （提示：可以得到整个玻璃面积白热化，并协助减缓年底的硅胶外套脱下，另一方面）。关闭火炬。 </li><li>关闭汞蒸馏。允许达到室温。关闭水。允许液氮慢慢蒸发。现在准备要放置在炉石英外套。 </李&gt; </ol> 5。于一炉加热反应混合物<ol><li>轻轻把铌管，中心石英夹克和超过热电偶直接放置。绝缘与玻璃棉炉的开口。关闭两天，在950°C炉和一组。 </li><li>项目建成后，使用玻璃切割圆锯（参见3.1），剖开石英管和删除铌容器。洗铌无任何碎片与水和丙酮在烘箱干燥。注入的drybox。 </li><li>铌管的两端用线切割钳切。轻轻地粉碎粗产品。优良的金属间化合物的前体是现在准备将带来了解决方案。 </li></ol> 6。溶解在乙二胺的易制毒化学<ol><li>掂量出一个试管到81毫克（0.1毫摩尔）4个9的K。添加搅拌酒吧。 </li><li> 2 - 吸取2.5毫升无水乙二胺（以前多金属钠蒸馏）5 - 10分钟，在室温下搅拌。一个鲜红的解决方案的形式。 </li><li>注：如果解决方案是搅太久，它会变成绿色，表明氧化集群。一个红色的解决方案所需的官能。 </li></ol> 7。 9集群与我反应锗3 SiC≡CSiMe的<ol><li>慢慢注射器，下拉明智，0.056毫升（0.25毫摩尔;超过2当量）小我3碳化硅≡CSiMe 3。含油层看到红色的群集解决方案上。随着反应的搅拌，溶液慢慢变成褐色。实现了四个小时的搅拌，直至蜂蜜棕色明确的解决方案。 </li><li>将试管离心15分钟。小心取出。插入一个干净的试管，玻璃纤维过滤器移液器（以前的包装，在180°C过夜干燥和抽成drybox）。 </li><li>小心进入过滤器的吸管吸取上清液，并允许进行筛选。明确蜂蜜棕色滤过TE是现在收集。 </li><li>预留0.1毫升滤液，以运行电喷雾质谱（ES-MS）的样本，以确认产品结晶前。 </li></ol> 8。运行ES-MS的反应液<ol><li>一个干净的1毫升汉密尔顿注射器，注射器活塞和PEEK管注入drybox前厅，至少30 - 45分钟。 </li><li>插入注射器注射器柱塞，并填写用无水乙二胺和分配到一个废物容器。清除注射器两次。 </li><li>无水乙二胺再次填补了注射器，并推动通过PEEK管。清除PEEK管两次以上。 PEEK管，现在充满了无水乙二胺。 </li><li>过滤葛9乙烯基解决方案填补空的注射器。 （提示：这可放置在充满氮气保鲜袋包运输，如果质谱仪的drybox的外）。衬托出drybox。 </li><li>地方注射器偷看在哈佛大学在10μL/ min的注射泵管。 PEEK管连接牢固电质谱仪。收集在1微团的Quattro-LC三重四极杆质谱仪负离子模式下的频谱（典型条件：100°C源温度125°C间脱溶剂温度，毛细管电压2.5千伏，30 - 65 V锥电压）或在布鲁克Microtof -II质谱仪（毛细管3800 V时，雾化器在0.6酒吧，脱溶剂温度在190°C间，毛细管出口在100 V，1200V的探测器）。 （注：样品高度空气和水分敏感，净化室15 - 60分钟才运行样本优先） </li><li>频谱将显示为官能集群同位素模式（见图1）。其余滤液在drybox现在可以结晶。 </li></ol> 9。 9 -二乙烯结晶锗离子与螯合剂<ol><li>吸管分为两个相等的等份剩余滤液在两个CLEAn测试管。标签试管C和D​​（作为一个例子）。预留。 </li><li>重达0.4毫摩尔无水18 - 冠-6（105毫克），到一个干净的试管，并加入8毫升的甲苯。拌匀至完全溶解。 </li><li>甲苯分层，方法：轻轻地吸取试管C和D​​（提示：尝试，以实现两个单独的阶段）上的4毫升这种解决方案对每个试管胶塞。预留试管架在一个不受干扰的结晶。 </li><li>分层甲苯，方法B：吸取18-crown-6/toluene解决方案4毫升到两个干净的试管标记E和D插入一个过滤器移液器（注：如果解决方案的需求将再次过滤）或定期将测试移液器管E和F吸取试管E和测试管D将C楼（注：这个反向分层分两个阶段，很快就会导致）解决方案，对每个试管胶塞。预留试管架在一个不受干扰的结晶。 </li><li>明亮的橙色块结晶于1- 3天。晶体单元，然后可以通过单晶X-射线衍射证实。 </li></ol> 10。检查单元细胞上的D8-衍射晶体<ol><li>填补与Paratone-N的油的塑料瓶，并允许所有的气泡被删除。完全脱气油前厅drybox，在真空条件下，过夜。带入的drybox。 </li><li>适用于2 - 3滴玻片。在石油和成结晶试管插入外套铲尖。从试管选择橙色水晶和沉浸在的Paratone-N的油。 （注意：确保所有空气敏感晶体在涂油）。使外drybox。 </li><li>用高分辨率显微镜，选择一个单一的晶体，并拖动到油直不锈钢探头幻灯片的边缘。 </li><li>小心取出水晶拖动到玻片边缘多余的油。安装Mitegen安装微循环和快速定位下冷水流（100K）的单晶配备CCD面积使用石墨单色器MoKα射线探测器布鲁克D8的APEX-II的衍射。 </li><li>确保良好的高角度的衍射和收购晶胞。 </li><li>比较和确认晶胞度（18 -冠-6）] 2 [葛9（HCCH 2）2]•EN，1，三斜晶系，磷-1，= 10.974（4），B = 14.3863（ 5），C = 16.2272（6）Å，α= 85.946（2），β= 71.136（2），γ= 89.264（2）°，V = 2412.21（15）3，Z = 53。 </li></ol> 11。代表结果阴离子簇的独特同位素模式，让他们可以很容易地检测在负离子模式（图1）。同样值得注意的是，减少单独收费的物种，除了与钾离子配对59是这种软电离技术的一个普遍现象。 晶体ST<html>与ructure有关债券的长度和角度[9戈（CH = CH 2）2] 2 - [K（18 -冠-6）] 2 [葛9（HCCH 2）2]•EN，1，可以看出在图2。 <img alt="图1" src="/files/ftp_upload/3532/3532fig1.jpg" /> 图1。ES -质谱（负离子模式）葛9集群与我3 SiC≡CSiMe的反应乙二胺解决方案。也低于实验分布的理论同位素分布。 （Sevov等。 无机化学，2007，46，10953。） <img alt="图2" src="/files/ftp_upload/3532/3532fig2.jpg" /> 图2。的视图[K（18 -冠-6）] 2 [葛9（HCCH 2）2]•恩，1。配色方案：<html> ullet1.jpg“/&gt; =戈， <img alt="子弹2" src="/files/ftp_upload/3532/3532bullet2.jpg" /> = C <img alt="子弹3" src="/files/ftp_upload/3532/3532bullet3.jpg" /> = H键长和角度：葛-C的1.961和1.950Å，= 1.318和1.316 nm，锗CC 123和127°。 （Sevov等。 无机化学，2007，46，10953。） <img alt="图3" src="/files/ftp_upload/3532/3532fig3.jpg" /> 图3示意图准备铌管的代表：（一）切割铌管;（二）在铌酸溶液清洗铌管;（三）用老虎钳交手卷曲和弯曲铌管。 <img alt="图4" src="/files/ftp_upload/3532/3532fig4.jpg" /> 图4铌管的准备：（一）电弧焊机图示意图;（二）在电弧焊机架交错铌管，（c）段铌管的焊接尖端。 / files/ftp_upload/3532/3532fig5.jpg“/&gt; 载入铌管的示意图： 图5。（一）（二）内drybox和焊前铌管：（一），（二）用老虎钳交手卷曲一个边缘（三）焊接一个边后，后， （四）后加载，然后焊接铌管封闭，开放铌管后采取的K 4个9的易制毒化学（V）。 <img alt="图6" src="/files/ftp_upload/3532/3532fig6.jpg" /> 图6。准备通过石英管的示意图玻璃吹（a）及（b）（一）大型和小型的石英管，（二）身体和颈部密封起来，（三）球头颈部（四）颈部及球头密封起来，（V），铌管密封石英管内，（六）后密封石英管。 <img alt="图7" src="/files/ftp_upload/3532/3532fig7.jpg" /> 图7。示意图Seali吴电熔硅胶管，高真空线（a）及（b）在铌管密封显示蚀刻石英管从铌酸溶液。 <img alt="图8" src="/files/ftp_upload/3532/3532fig8.jpg" /> 图8。放置在炉加载的熔融石英管的示意图。 <img alt="图9" src="/files/ftp_upload/3532/3532fig9.jpg" /> 图9。示意图反应钾K 4个9 3碳化硅≡3 CSiMe内的drybox（一）（一）未开封的铌管，（二）（三）剪钳，切割铌管的一个边缘（ IV）粉碎及易制毒化学（二）（一）易制毒化学在乙二胺解散，（ii）紧随我之后3碳化硅≡CSiMe 3添加（试管看到墙壁上的油性液滴）。 <img alt="图10" src="/files/ftp_upload/3532/3532fig10.jpg" /> 连接古雷10。示意图表示，ES-MS的反应液中运行（一）质谱仪在烘干箱中准备的注射器（二）布鲁克Microtof-II。 <img alt="图11" src="/files/ftp_upload/3532/3532fig11.jpg" /> 图11。结晶戈9-乙烯基螯合剂（一）扭转分层及（b）几个小时后的示意图。 <img alt="图12" src="/files/ftp_upload/3532/3532fig12.jpg" /> 图12检查单细胞上的D8-衍射晶体示意图：（一）选择在显微镜下的晶体和（b）收集单元。

没有利益冲突的声明。

这是重要的清洁以及部分氧化铌管。然而，如果留在管铌清洗液的时间过长，将严重危及管的厚度。因此，10 - 15秒，是势在必行，管，应该是在结束（图3）非常有光泽。后密封石英外套管内，他们应与铌稀溶液清洗了一遍。这将导致轻度欢腾，清洁氧化铌管焊接或玻璃吹制过程中发生的任何地区。但是，要小心不要离开太久溶液蚀刻玻璃（图7B）。 重要的是要确保碱金属?...

<table border="1"><tbody><tr><td> 试剂名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论 </td></tr><tr><td> D8的，X射线衍射仪</td><td>布鲁克</td><td>布鲁克的APEX II </td><td></td></tr><tr><td>电质谱仪</td><td>布鲁克</td><td> microtof-II </td><td></td></tr><tr><td>电质谱仪</td><td>微团</td><td> QUATTRO-LC </td><td>三，quadropole </td></tr><tr><td> drybox </td><td>创新科技</td><td>的S-1的M-DL </td><td> IT-系统1模型</td></tr><tr><td>惰性气体/真空屏蔽 弧焊安排</td><td> <a href="http://www.ldsvacuum.com" target="_blank">LDS的真空产品</a> </td><td>特别订购</td><td></td></tr><tr><td>电弧焊机电源</td><td>米勒&lt;/ TD&gt; <td> maxstar-91 </td><td></td></tr><tr><td>焊接橡胶手套</td><td> Home Depot的</td><td> KH643 </td><td></td></tr><tr><td>电动雕刻机</td><td>伯吉斯产品</td><td> 74 </td><td>振动雕刻刀</td></tr><tr><td>圆形玻璃锯</td><td>皮斯托留斯机械有限责任公司</td><td> GC-12-B的</td><td></td></tr><tr><td>管式炉</td><td>林德伯格/蓝M </td><td> TF55035 </td><td> minimite实验室管式炉，Moldatherm（1100°C） </td></tr><tr><td>玻璃干燥箱</td><td> Fisher Scientific则</td><td> 13-247-650G </td><td></td></tr><tr><td>高真空汞舒伦克线</td><td>特别订购</td><td>大学的巴黎圣母院</td><td>替代：E050/60爱德华兹; VWR International的猫。 EVB302-07-110号</td></tr><tr><td>大火炬</td><td>胜利者</td><td> JT100C &lt;/ TD&gt; <td>焊枪，提示：维克多5 - WJ </td></tr><tr><td>小火炬</td><td> veriflo有限公司</td><td> 3A </td><td>吹塑管</td></tr><tr><td>特斯拉线圈</td><td> VWR国际</td><td> KT691550-0000 </td><td>检漏仪</td></tr><tr><td>搅拌器/热板</td><td> VWR国际</td><td> 12620-970 </td><td>厂商VWR热板STR DY-DUAL120V </td></tr><tr><td>平衡</td><td>丹佛仪器有限公司</td><td> 100A </td><td> XE系列</td></tr><tr><td>离心分离</td><td>长波科学</td><td> E8C-08AV-1501 </td><td>变速</td></tr><tr><td>石墨铰刀，（燃烧） </td><td> ABR的意象，公司</td><td> 850-523生鲜</td><td>吹玻璃开孔和燃烧边缘</td></tr><tr><td>前锋</td><td> Fisher Scientific则</td><td> 12-007 </td><td></td></tr><tr><td>虎钳，夹具</td><td>家仓库</td><td> 0902L3SM </td><td></td></tr><tr><td>管刀</td><td> Home Depot的</td><td> 32820 </td><td></td></tr><tr><td>钢丝钳</td><td> Home Depot的</td><td> 437 </td><td></td></tr><tr><td>塑料烧杯</td><td> VWR国际</td><td> 13890-046 </td><td></td></tr><tr><td>量筒</td><td> VWR国际</td><td> 65000-006 </td><td>小心，高频蚀刻玻璃（如果使用玻璃） </td></tr><tr><td>大型塑料瓶</td><td> VWR国际</td><td> 16128-542 </td><td></td></tr><tr><td> 13×100试管</td><td> VWR国际</td><td> 47729-572 </td><td> 13X100 CS1000的培养管</td></tr><tr><td>实验室（橡胶）塞</td><td> Sigma公司</td><td> Z164437-100EA </td><td>大小00 </td></tr><tr><td> ţEST-试管架</td><td> VWR国际</td><td> 60196-702 </td><td> 10-13毫米管外径</td></tr><tr><td>搅拌的酒吧</td><td> StirBars.com /大科技公司</td><td> SBM-0803-麦克风</td><td>聚四氟乙烯8x3毫米微</td></tr><tr><td>玻璃滴管</td><td> VWR国际</td><td> 14673-043 </td><td>厂商VWR吸取巴斯德9英寸CS1000的</td></tr><tr><td>橡胶灯泡</td><td> VWR国际</td><td> 56311-062 </td><td>乳胶，薄壁</td></tr><tr><td>玻璃棉</td><td> Unifrax的我LLC </td><td> 6048 </td><td> fiberfrax散装纤维绝缘材料，陶瓷纤维</td></tr><tr><td>载玻片</td><td> VWR国际</td><td> 16004-422 </td><td> 75x25x1mm，显微镜载玻片</td></tr><tr><td> paratone-N的石油</td><td>汉普顿研究</td><td> parabar 10312 </td><td>称为：Paratone-N Paratone，8277，润英V8512 </td></tr><tr><td>高真空硅脂</td><td> VWR国际</td><td> 59344-055 </td><td>道康宁</td></tr><tr><td>液氮</td><td>大学巴黎圣母院</td><td></td><td></td></tr><tr><td>氩气瓶</td><td>普莱克斯公司经销公司</td><td> TARGHP </td><td></td></tr><tr><td>氮气气瓶</td><td>普莱克斯公司经销公司</td><td> QNITPP </td><td></td></tr><tr><td>氧气钢瓶</td><td>普莱克斯公司经销公司</td><td>加时赛</td><td> 337比照共青团</td></tr><tr><td>氢气钢瓶</td><td>普莱克斯公司经销公司</td><td>港币</td><td> 195比照共青团</td></tr><tr><td>丙烷气瓶/源</td><td>大学巴黎圣母院</td><td>北达科</td><td></td></tr><tr><td>石英管，LG </td><td>石英科技公司</td><td> 100020B </td><td> 20毫米内径x 22毫米外径x 48“清晰的融合夸脱油管</td></tr><tr><td>石英管，马里兰州</td><td>石英科技公司</td><td> 100007B </td><td>清除熔融石英管，7毫米ID x 9mm的外径×48“ </td></tr><tr><td>圆底石英关节的</td><td>石英科技公司</td><td> 6160189B </td><td>球节</td></tr><tr><td>石英安全眼镜</td><td>甘文维仪器</td><td> 11-1127 </td><td> waleapparatus.com </td></tr><tr><td> Pyrex的安全眼镜</td><td>甘文维仪器</td><td> 11-2125-B3 </td><td>对于明确和彩色高硼硅玻璃</td></tr><tr><td>吹软管套件</td><td>玻璃屋</td><td> BH020 </td><td> glasshousesupply.com </td></tr><tr><td>铌管</td><td>陕西托尼金属有限公司</td><td>铌管，50英尺</td><td>无缝铌管外径：0.375（±0.005英寸）。 壁厚：0.02（±0应退火0.003）英寸铌。 </td></tr><tr><td>为群众SPECT PEEK入门套件</td><td>水域</td><td> PSL613321 </td><td>的PEEK（聚醚醚酮）管，螺母，垫圈，适合</td></tr><tr><td>质量SPECT针集</td><td> VWR国际</td><td> 60373-992 </td><td>汉密尔顿制造商（81165） </td></tr><tr><td>以H 2 SO 4 </td><td> VWR国际</td><td> BDH3072-2.5LG </td><td> ACS级</td></tr><tr><td> 硝酸 </td><td> VWR国际</td><td> BDH3046-2.5LPC </td><td> ACS级</td></tr><tr><td>高频</td><td> VWR国际</td><td> BDH3040-500MLP </td><td> ACS级</td></tr><tr><td>蒸馏水</td><td>大学巴黎圣母院</td><td>北达科</td><td></td></tr><tr><td>丙酮</td><td> VWR国际</td><td> BDH1101-4LP </td><td></td></tr><tr><td>乙二胺</td><td> VWR国际</td><td> AAA12132-0F </td><td> 2.5升99％ </td></tr><tr><td>甲苯</td><td> VWR国际</td><td> 200004-418 </td><td> 99.8％，无水</td></tr><tr><td>水星</td><td> STREM化工公司</td><td> 93-8046 </td><td></td></tr><tr><td>钾（K）金属</td><td> STREM化工公司</td><td> 19-1989 </td><td>在氩气密封玻璃安瓿</td></tr><tr><td>锗（Ge）粉</td><td> VWR国际</td><td> AA10190-18 </td><td>胚芽PWR -100目99.999％50G </td></tr><tr><td> bistrimetylsilylacetylene， （ME 3 SiC≡CCSiMe的） </td><td>菲舍尔科学</td><td> AC182010100 </td><td></td></tr><tr><td> 18 - 冠-6 （1,4,7,10,13,16-Hexaoxacyclooctadecane） </td><td> VWR国际</td><td> 200001-954 </td><td> 99％，25克</td></tr><tr><td> 2,2,2-crypt加密 （4,7,13,16,21,24-Hexaoxa-1，10二氮杂双环[8.8.8]二十六烷） </td><td> Sigma Aldrich公司</td><td> 291110-1G </td><td> 98％ </td></tr></tbody></table>

synthesis of nine-atom deltahedral zintl ions of germanium and their functionalization with organic groups

虽然1890年代末和1930年初的Zintl离子之间的日期最初的研究，他们并没有结构上的特点，直到多年以后。1,2它们的氧化还原化学是更年轻的，只是10岁，但尽管这短暂的历史这些离子deltahedral的集群北京时间9 N-（C =硅，锗，锡，铅，N = 2，3，4）。3-6已经显示出有趣的和多样化的反应，并已在迅速发展和令人振奋的新化学前沿的著名的里程碑9月 4锗的氧化耦合-低聚物和无限链，7-19金属化，14-16,20-25集群过渡金属有机碎片，26-34过渡金属原子插入在中心封顶有时被封盖和齐聚，除了主族有机金属碎片外保税取代35-47结合的产业集群，48-50和官能与各种有机残留物与有机卤化物和炔烃的反应。51-58 这后者的有机碎片直接连接集群的发展开辟了一个新的领域，即的有机Zintl化学，为进一步合成的探索，可能是肥沃的，它是一步一步由锗乙烯基合成过程此处描述的集群。最初的步骤概述合成革9 K的4间易制毒化学从戈9 4 -簇后在溶液中提取。这涉及到熔石英玻璃吹，铌容器弧焊，高度空气敏感材料的处理，在手套箱。空气敏感K戈9，然后在框中乙二胺溶解，然后跟我反应3 SiC≡CSiMe的alkenylated。反应，其次是电trospray质谱，而最终的解决方案是用获得单晶包含官能集群[H 2 C = CH戈9-CH = CH 2] 2 - 。用于此目的的解决方案是离心，过滤，并仔细分层与18 - 冠-6的甲苯溶液。离开几天原状，这样层次的解决方案产生的橙色[K（18 -冠-6）] 2 [葛9（HCCH 2）2]•EN由单晶X-射线衍射特征结晶块。 过程中突出反应技术标准，工作，和对官能deltahedral Zintl集群分析。它希望，这将有助于对这些化合物在大进一步发展和社会的理解。

Watch this Scientific Journal Video about Synthesis of Nine-atom Deltahedral Zintl Ions of Germanium and their Functionalization with Organic Groups at JoVE.com

Synthesis of Nine-atom Deltahedral Zintl Ions of Germanium and their Functionalization with Organic Groups

我们提出的K高温金属间化合物的前体的合成<sub&gt; 4</sub&gt; GE<sub&gt; 9</sub&gt;，他们在解散乙二胺形成GE<sub&gt; 9</sub<sup&gt; 4 -</sup&gt; deltahedral Zintl离子，炔集群的反应形成有机Zintl离子。后者的特点是通过电质谱解决方案，并通过单晶X-射线衍射固态。

九的原子Deltahedral Zintl离子锗和他们的官能合成有机基团

Although the first studies of Zintl ions date between the late 1890's and early 1930's they were not structurally characterized until many years later.1,2 ...

synthesis-nine-atom-deltahedral-zintl-ions-germanium-their

Research

JoVE Journal

Biology

13.9K 次观看.  University of Notre Dame. 我们提出的K高温金属间化合物的前体的合成 4 GE 9，他们在解散乙二胺形成GE 9

视频: Synthesis of Nine-atom Deltahedral Zintl Ions of Germanium and their Functionalization with Organic Groups

Staining of Proteins in Gels with Coomassie G-250 without Organic Solvent and Acetic Acid

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents (Methanol, Ethanol or 2-Propanol) and acetic acid are used for fixation, staining and destaining of proteins in a gel after SDS-PAGE. To speed up the procedure, heating the staining solution in the microwave oven for a short time is frequently used. This usually results in evaporation of toxic or hazardous Methanol, Ethanol or 2-Propanol and a strong smell of acetic acid in the lab which should be avoided due to safety considerations. In a protocol originally published in two patent applications by E.M. Wondrak (US2001046709 (A1), US6319720 (B1)), an alternative composition of the staining solution is described in which no organic solvent or acid is used. The CBB is dissolved in bidistilled water (60-80mg of CBB G-250 per liter) and 35 mM HCl is added as the only other compound in the staining solution. The CBB staning of the gel is done after SDS-PAGE and thorough washing of the gel in bidistilled water. By heating the gel during the washing and staining steps, the process can be finished faster and no toxic or hazardous compunds are evaporating. The staining of proteins occurs already within 1 minute after heating the gel in staining solution and is fully developed after 15-30 min with a slightly blue background that is destained completely by prolonged washing of the stained gel in bidistilled water, without affecting the stained protein bands.

A short protocol for protein staining with Coomassie Brilliant Blue (CBB) G-250 in polyacrylamide gels is described without using organic solvents or acetic acid as in the classical staining procedures with CBB.

无有机溶剂和醋酸，蛋白质与考马斯亮蓝G - 250凝胶染色

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents ...

科研

生物学

Profiling of Methyltransferases and Other S-adenosyl-L-homocysteine-binding Proteins by Capture Compound Mass Spectrometry (CCMS)

There is a variety of approaches to reduce the complexity of the proteome on the basis of functional small molecule-protein interactions such as affinity chromatography 1 or Activity Based Protein Profiling 2. Trifunctional Capture Compounds (CCs, Figure 1A) 3 are the basis for a generic approach, in which the initial equilibrium-driven interaction between a small molecule probe (the selectivity function, here S-adenosyl-L-homocysteine, SAH, Figure 1A) and target proteins is irreversibly fixed upon photo-crosslinking between an independent photo-activable reactivity function (here a phenylazide) of the CC and the surface of the target proteins. The sorting function (here biotin) serves to isolate the CC - protein conjugates from complex biological mixtures with the help of a solid phase (here streptavidin magnetic beads). Two configurations of the experiments are possible: "off-bead" 4 or the presently described "on-bead" configuration (Figure 1B). The selectivity function may be virtually any small molecule of interest (substrates, inhibitors, drug molecules).
S-Adenosyl-L-methionine (SAM, Figure 1A) is probably, second to ATP, the most widely used cofactor in nature 5, 6. It is used as the major methyl group donor in all living organisms with the chemical reaction being catalyzed by SAM-dependent methyltransferases (MTases), which methylate DNA 7, RNA 8, proteins 9, or small molecules 10. Given the crucial role of methylation reactions in diverse physiological scenarios (gene regulation, epigenetics, metabolism), the profiling of MTases can be expected to become of similar importance in functional proteomics as the profiling of kinases. Analytical tools for their profiling, however, have not been available. We recently introduced a CC with SAH as selectivity group to fill this technological gap (Figure 1A).
SAH, the product of SAM after methyl transfer, is a known general MTase product inhibitor 11. For this reason and because the natural cofactor SAM is used by further enzymes transferring other parts of the cofactor or initiating radical reactions as well as because of its chemical instability 12, SAH is an ideal selectivity function for a CC to target MTases. Here, we report the utility of the SAH-CC and CCMS by profiling MTases and other SAH-binding proteins from the strain DH5&alpha; of Escherichia coli (E. coli), one of the best-characterized prokaryotes, which has served as the preferred model organism in countless biochemical, biological, and biotechnological studies. Photo-activated crosslinking enhances yield and sensitivity of the experiment, and the specificity can be readily tested for in competition experiments using an excess of free SAH.

Profiling of Methyltransferases and Other S-adenosyl-L-homocysteine-binding Proteins by Capture Compound Mass Spectrometry CCMS

Capture Compounds are trifunctional small molecules to reduce the complexity of the proteome by functional reversible small molecule-protein interaction followed by photo-crosslinking and purification. Here we use a Capture Compound with S-adenosyl-L-homocysteine-binding as selectivity function to isolate methyltransferases from an Escherichia coli whole cell lysate and identify them by MS.

分析甲基化和其他 S腺苷 L</sub捕捉复合质谱（CCMS）>同型半胱氨酸结合蛋白

There is a variety of approaches to reduce the complexity of the proteome on the basis of functional small molecule-protein interactions such as affinity ...

High-Throughput Measurement and Classification of Organic P in Environmental Samples

Many types of organic phosphorus (P) molecules exist in environmental samples1. Traditional P measurements do not detect these organic P compounds since they do not react with colorimetric reagents2,3. Enzymatic hydrolysis (EH) is an emerging method for accurately characterizing organic P forms in environmental samples4,5. This method is only trumped in accuracy by Phosphorus-31 Nuclear Magnetic Resonance Spectroscopy (31P-NMR) -a method that is expensive and requires specialized technical training6. We have adapted an enzymatic hydrolysis method capable of measuring three classes of phosphorus (monoester P, diester P and inorganic P) to a microplate reader system7. This method provides researchers with a fast, accurate, affordable and user-friendly means to measure P species in soils, sediments, manures and, if concentrated, aquatic samples. This is the only high-throughput method for measuring the forms and enzyme-lability of organic P that can be performed in a standard laboratory. The resulting data provides insight to scientists studying system nutrient content and eutrophication potential.

This protocol describes a high-throughput method of enzymatic hydrolysis that utilizes a microplate reader to measure and classify soil phosphorus as P monoesters, P diesters and inorganic P. Up to 96 samples can be measured at one time in a standard laboratory.

高通量测量和环境样品中的有机磷分类

Many types of organic phosphorus (P) molecules exist in environmental samples1.  Traditional P measurements do not detect these organic P compounds since ...

A Chemical Screening Procedure for Glucocorticoid Signaling with a Zebrafish Larva Luciferase Reporter System

Glucocorticoid stress hormones and their artificial derivatives are widely used drugs to treat inflammation, but long-term treatment with glucocorticoids can lead to severe side effects. Test systems are needed to search for novel compounds influencing glucocorticoid signaling in vivo or to determine unwanted effects of compounds on the glucocorticoid signaling pathway. We have established a transgenic zebrafish assay which allows the measurement of glucocorticoid signaling activity in vivo and in real-time, the GRIZLY assay (Glucocorticoid Responsive In vivo Zebrafish Luciferase activitY). The luciferase-based assay detects effects on glucocorticoid signaling with high sensitivity and specificity, including effects by compounds that require metabolization or affect endogenous glucocorticoid production. We present here a detailed protocol for conducting chemical screens with this assay. We describe data acquisition, normalization, and analysis, placing a focus on quality control and data visualization. The assay provides a simple, time-resolved, and quantitative readout. It can be operated as a stand-alone platform, but is also easily integrated into high-throughput screening workflows. It furthermore allows for many applications beyond chemical screening, such as environmental monitoring of endocrine disruptors or stress research.

We describe the procedure and data analysis of a chemical screening system for glucocorticoid stress hormone signaling using zebrafish larvae: the Glucocorticoid Responsive In vivo Zebrafish Luciferase activitY (GRIZLY) assay. The assay sensitively and specifically detects effects on glucocorticoid signaling by compounds that require metabolization or affect endogenous glucocorticoid production.

对糖皮质激素信号A化学筛选程序与斑马鱼幼虫的荧光素酶报告系统

Glucocorticoid stress hormones and their artificial derivatives are widely used drugs to treat inflammation, but long-term treatment with glucocorticoids ...

Rapid Synthesis and Screening of Chemically Activated Transcription Factors with GFP-based Reporters

Synthetic biology aims to rationally design and build synthetic circuits with desired quantitative properties, as well as provide tools to interrogate the structure of native control circuits. In both cases, the ability to program gene expression in a rapid and tunable fashion, with no off-target effects, can be useful. We have constructed yeast strains containing the ACT1 promoter upstream of a URA3 cassette followed by the ligand-binding domain of the human estrogen receptor and VP16. By transforming this strain with a linear PCR product containing a DNA binding domain and selecting against the presence of URA3, a constitutively expressed artificial transcription factor (ATF) can be generated by homologous recombination. ATFs engineered in this fashion can activate a unique target gene in the presence of inducer, thereby eliminating both the off-target activation and nonphysiological growth conditions found with commonly used conditional gene expression systems. A simple method for the rapid construction of GFP reporter plasmids that respond specifically to a native or artificial transcription factor of interest is also provided.

This protocol describes an experimental procedure for the rapid construction of artificial transcription factors (ATFs) with cognate GFP reporters&#160;and quantification of the ATFs ability to stimulate GFP expression via flow cytometry.

快速合成及其与绿色荧光蛋白为基础的记者化学激活的转录因子筛选

Synthetic biology aims to rationally design and build synthetic circuits with desired quantitative properties, as well as provide tools to interrogate the ...

Non-chromatographic Purification of Recombinant Elastin-like Polypeptides and their Fusions with Peptides and Proteins from Escherichia coli

Elastin-like polypeptides are repetitive biopolymers that exhibit a lower critical solution temperature phase transition behavior, existing as soluble unimers below a characteristic transition temperature and aggregating into micron-scale coacervates above their transition temperature. The design of elastin-like polypeptides at the genetic level permits precise control of their sequence and length, which dictates their thermal properties. Elastin-like polypeptides are used in a variety of applications including biosensing, tissue engineering, and drug delivery, where the transition temperature and biopolymer architecture of the ELP can be tuned for the specific application of interest. Furthermore, the lower critical solution temperature phase transition behavior of elastin-like polypeptides allows their purification by their thermal response, such that their selective coacervation and resolubilization allows the removal of both soluble and insoluble contaminants following expression in Escherichia coli. This approach can be used for the purification of elastin-like polypeptides alone or as a purification tool for peptide or protein fusions where recombinant peptides or proteins genetically appended to elastin-like polypeptide tags can be purified without chromatography. This protocol describes the purification of elastin-like polypeptides and their peptide or protein fusions and discusses basic characterization techniques to assess the thermal behavior of pure elastin-like polypeptide products.

Non-chromatographic Purification of Recombinant Elastin-like Polypeptides and their Fusions with Peptides and Proteins from Escherichia coli

Elastin-like polypeptides are stimulus-responsive biopolymers with applications ranging from recombinant protein purification to drug delivery. This protocol describes the purification and characterization of elastin-like polypeptides and their peptide or protein fusions from Escherichia coli using their lower critical solution temperature phase transition behavior as a simple alternative to chromatography.

重组弹性蛋白样多肽及其融合物与从肽和蛋白质非色谱纯化<em&gt;大肠杆菌</em

Elastin-like polypeptides are repetitive biopolymers that exhibit a lower critical solution temperature phase transition behavior, existing as soluble ...

Sequence-specific Labeling of Nucleic Acids and Proteins with Methyltransferases and Cofactor Analogues

S-Adenosyl-l-methionine (AdoMet or SAM)-dependent methyltransferases (MTase) catalyze the transfer of the activated methyl group from AdoMet to specific positions in DNA, RNA, proteins and small biomolecules. This natural methylation reaction can be expanded to a wide variety of alkylation reactions using synthetic cofactor analogues. Replacement of the reactive sulfonium center of AdoMet with an aziridine ring leads to cofactors which can be coupled with DNA by various DNA MTases. These aziridine cofactors can be equipped with reporter groups at different positions of the adenine moiety and used for Sequence-specific Methyltransferase-Induced Labeling of DNA (SMILing DNA). As a typical example we give a protocol for biotinylation of pBR322 plasmid DNA at the 5&#8217;-ATCGAT-3&#8217; sequence with the DNA MTase M.BseCI and the aziridine cofactor 6BAz in one step. Extension of the activated methyl group with unsaturated alkyl groups results in another class of AdoMet analogues which are used for methyltransferase-directed Transfer of Activated Groups (mTAG). Since the extended side chains are activated by the sulfonium center and the unsaturated bond, these cofactors are called double-activated AdoMet analogues. These analogues not only function as cofactors for DNA MTases, like the aziridine cofactors, but also for RNA, protein and small molecule MTases. They are typically used for enzymatic modification of MTase substrates with unique functional groups which are labeled with reporter groups in a second chemical step. This is exemplified in a protocol for fluorescence labeling of histone H3 protein. A small propargyl group is transferred from the cofactor analogue SeAdoYn to the protein by the histone H3 lysine 4 (H3K4) MTase Set7/9 followed by click labeling of the alkynylated histone H3 with TAMRA azide. MTase-mediated labeling with cofactor analogues is an enabling technology for many exciting applications including identification and functional study of MTase substrates as well as DNA genotyping and methylation detection.

DNA and proteins are sequence-specifically labeled with affinity or fluorescent reporter groups using DNA or protein methyltransferases and synthetic cofactor analogues. Depending on the cofactor specificity of the enzymes, aziridine or double activated cofactor analogues are employed for one- or two-step labeling.

核酸，蛋白质与甲基转移和辅酶类似物序列特异性标签

S-Adenosyl-l-methionine (AdoMet or SAM)-dependent methyltransferases (MTase) catalyze the transfer of the activated methyl group from AdoMet to specific ...

Detection of Intracellular Gene Expression in Live Cells of Murine, Human and Porcine Origin Using Fluorescence-labeled Nanoparticles

The reprogramming of somatic cells to induced pluripotent stem cells (iPS) has successfully been performed in different mammalian species including mouse, rat, human, pig and others. The verification of iPS clones mainly relies on the detection of the endogenous expression of different pluripotency genes. These genes mostly represent transcription factors which are located in the cell nucleus. Traditionally, the proof of their endogenous expression is supplied by immunohistochemical staining after fixation of the cells. This approach requires replicate cultures of each clone at this early stage to preserve validated clones for further experiments. The present protocol describes an approach with gene-specific nanoparticles which allows the evaluation of intracellular gene expression directly in live cells by fluorescence. The nanoparticles consist of a central gold particle coupled to a capture strand carrying a sequence complementary to the target mRNA as well as a quenched reporter strand. These nanoparticles are actively endocytosed and the target mRNA displaces the reporter strand which then start to fluoresce. Therefore, specific target gene expression can be detected directly under the microscope. In addition, the emitted fluorescence allows the identification, isolation and enrichment of cells expressing a specific gene by flow cytometry. This method can be applied directly to live cells in culture without any manipulation of the target cells.

This manuscript describes a novel technique which allows the detection of intracellular gene expression after endocytosis of fluorescence-labeled nanoparticles directly in live cells. The method does not require manipulation of the cells and is not restricted with respect to the target gene or species.

利用荧光标记的纳米粒子检测的小鼠，人与猪源活细胞内的基因表达

The reprogramming of somatic cells to induced pluripotent stem cells (iPS) has successfully been performed in different mammalian species including mouse, ...

Synthesis of Wavelength-shifting DNA Hybridization Probes by Using Photostable Cyanine Dyes

In this protocol, we demonstrate a method for the synthesis of 2&#39;-alkyne modified deoxyribonucleic acid (DNA) strands by automated solid phase synthesis using standard phosphoramidite chemistry. Oligonucleotides are post-synthetically labeled by two new photostable cyanine dyes using copper-catalyzed click-chemistry. The synthesis of both donor and acceptor dye is described and is performed in three consecutive steps. With the DNA as the surrounding architecture, these two dyes undergo an energy transfer when they are brought into close proximity by hybridization. Therefore, annealing of two single stranded DNA strands is visualized by a change of fluorescence color. This color change is characterized by fluorescence spectroscopy but can also be directly observed by using a handheld ultraviolet (UV) lamp. The concept of a dual fluorescence color readout makes these oligonucleotide probes excellent tools for molecular imaging especially when the described photostable dyes are used. Thereby, photobleaching of the imaging probes is prevented, and biological processes can be observed in real time for a longer time period.

Photostable cyanine dyes are attached to oligonucleotides to monitor hybridization by energy transfer.

通过使用光稳定菁染料波长移的DNA杂交探针的合成

In this protocol, we demonstrate a method for the synthesis of 2&#39;-alkyne modified deoxyribonucleic acid (DNA) strands by automated solid phase ...

Isolation of Mouse Epidermal Keratinocytes and Their In Vitro Clonogenic Culture

The protocol described here is a reliable method of harvesting primary keratinocytes from adult female mice (54 &#177; 2 days old) yielding approximately 30 x 106 viable cells per mouse. Primary adult mouse keratinocytes are harvested from the dorsal skin of female mice. Male mice (~6 weeks old) can be used for keratinocyte harvesting depending on the requirements of the experiment. Euthanized mice are shaved and sterilized with serial washes in povidone iodine and ethanol solutions (70% alcohol). After disinfecting the mice, the dorsal skin is removed and the subcutaneous fat and muscle are removed with a scalpel and discarded. The skins are cut into small pieces and treated with a mild, low temperature trypsinization to detach the lower dermis from the epidermis. The scraped epidermises are stirred at low speed, filtered to remove the hairs, counted, and re-suspended in culture medium. This method provides an excellent single cell suspension of highly culturable cells for many downstream applications.

The goal of this protocol is to isolate epidermal keratinocytes from the dorsal skin of adult mice for a variety of downstream applications such as molecular biology, biochemistry, fluorescence activated cell sorting, and primary in vitro uses (e.g., clonogenic keratinocytes).

小鼠表皮角质细胞的分离及其体外克隆培养物

The protocol described here is a reliable method of harvesting primary keratinocytes from adult female mice (54 &#177; 2 days old) yielding approximately ...