该莱夫科夫斯基家庭基金会/ Liz和埃里克·莱夫科夫斯基创新研究奖，以AV我们想 - 这里介绍的项目是由美国国立卫生研究院从/ NCI（NCI-R01CA182506-01A1）的资助，以及由罗伯特·卢里综合癌症中心的支持要感谢批判性阅读和编辑这篇稿子的实验室成员（卡罗尔·奥卡拉汉和伊丽莎白安妮·韦恩机场）。

1. 在体外试验脱乙酰<ol><li> SIRT2纯化<ol><li>准备在8ml含10％FBS和抗生素的培养，并在37℃，5％CO 2的组织培养箱中生长至约70％汇合，第二天的HEK 293T细胞的10cm的培养皿。 </li><li>第二天，共转染4微克PCDH-普罗-GFP-SIRT2-标志（在我们的实验室制成慢病毒），4微克pCMV- dR8.2 DVPR（包装载体）和0.5微克的pCMV-VSV-G（包络线矢量） 18使用聚乙烯亚胺（PEI）在3微升的PEI /微克DNA的一比值。 </li><li>经过12小时更换介质。 </li><li>后36-48小时收集上清。 </li><li>离心1000 XG 5分钟，4℃清除杂物。 </li><li>通过0.22微米的过滤器过滤后，使SIRT2慢病毒的1ml等份，并保持在-80℃。 </li><li>在冰上解冻SIRT2慢病毒（对病毒储存在-80℃下是很重要的，当不使用病毒）。 </li><li>感染80-90％汇合的HEK 293T细胞的6孔平板，在8微克/毫升聚凝胺的具有1毫升的慢病毒。 </li><li>放置在37℃培养箱中病毒感染的细胞，直到第二天（24小时）。 </li><li> 24小时后更换培养基。 </li><li>感染后48小时，通过在荧光显微镜下检测GFP阳性细胞检查感染效率。 </li><li>如果感染效率是好的（至少40-50％感染的细胞），含有2微克/毫升嘌呤霉素的培养基中过表达的细胞，选择稳定SIRT2更换介质。 注意:这大约需要2周，并且将培养基每3-4天更换一次。非感染细胞的死亡在这个选择期间，只感染细胞的慢病毒SIRT2增长。 </li><li>在10％SDS-PAGE凝胶上运行40微克总蛋白后:（1,000稀释1）在开始PURIF之前通过Western印迹使用抗Flag抗体分析Flag标记SIRT2的表达ication过程（推荐）19,20。 </li><li>净化活性SIRT2，分裂细胞以1:3的比例通过胰蛋白酶消化，以便有HEK 293T细胞稳定过表达的Flag-SIRT2的足够菜（十10厘米菜将使随后的实验够蛋白的纯化）。 <ol><li>到trypsinize的细胞，除去培养基，并通过漂洗细胞，用无菌DPBS消除残余的血清。慢慢加入2.5 0.25％胰蛋白酶-EDTA溶液中，以覆盖细胞单层，在室温下孵育30-45秒。除去胰蛋白酶-EDTA溶液后，孵育在37℃，直至细胞开始分离。再加入培养液和细胞转移到新菜。 </li></ol></li><li>除去培养基，在4℃下以1000 xg离心在PBS洗涤细胞两次，并收集通过胰蛋白酶的细胞，然后离心10分钟。 </li><li>于500μl裂解缓冲液A（20mM的HEPES pH值7.9，18毫米氯化钾，0.2mM的EGTA，1.5mM的MgCl 2的裂解细胞沉淀，20％甘油，0.1％NP-40），其包括蛋白酶抑制剂鸡尾酒和下恒定旋转1μM的TSA的30分钟，在4℃。 </li><li>离心机在14000 xg离心在4℃下15分钟，并转移上清液至新管。 </li><li>使用缀合，如下所述琼脂糖珠粒的抗Flag抗体进行免疫沉淀。 <ol><li>添加200-300微升偶联至琼脂糖珠的蛋白质裂解物（10-20毫克总蛋白）抗Flag抗体的。 </li><li>在4℃下以恒定的搅拌温育过夜。 </li><li>收集离心免疫沉淀在1000 xg离心在4℃下5分钟。 </li><li>洗涤沉淀5次，用10ml裂解缓冲液A以1000 xg离心在4℃下5分钟。每次洗涤后，沉淀珠子和替换用缓冲液A将上清液</li></ol></li><li>制备1×FLAG肽溶液（包括蛋白酶抑制剂鸡尾酒和1μM的TSA在PBS中）。 </li><li>加入500μl1X标志肽类的Ë溶液到琼脂糖珠，并在4℃下恒定旋转温育30分钟。 </li><li>收集离心上清在6000 xg离心在4℃下2分钟。 </li><li>重复前两个步骤。 </li><li>通过使用过滤管和离心15,000 XG 1分钟，4℃卸下上清珠。 </li><li>浓缩洗脱样品，直到最终蛋白质浓度为1微克/微升使用超滤膜。 </li><li>通过使用洗脱样品的1微克进行电泳检查纯化过程。 </li><li>用市售的染色液染色凝胶来确认SIRT2纯化。 </li><li>保持纯净SIRT2在-80℃。 </li></ol></li><li>乙酰化的蛋白质 - 底物的纯化<ol><li>准备好10厘米×10厘米的培养皿与HEK 293T细胞为约70％汇合的第二天。 </li><li>第二天，共转染细胞与标记的5微克标记质粒载体expressin克蛋白质 - 底物以及5微克特定乙酰转移可在3微升的PEI /微克DNA的一比率乙酰使用的PEI的蛋白质。 注意:如果特定乙酰转移是未知的，共转染与已知的组蛋白乙酰基转移酶（帽）如P300，CBP，GCN5，Tip60的和PCAF的混合物。 </li><li>经过12小时更换培养基。 </li><li>当天裂解细胞前，治疗用1μMTSA和2mM烟酰胺（NAM）过夜的细胞通过更换用含TSA / NAM通过抑制脱乙酰酶最大化蛋白质 - 底物的乙酰化水平的介质的培养基中。 </li><li>转染后48小时，去除培养基，在PBS中通过胰蛋白酶消化，随后离心以1000 xg离心在4℃下洗涤细胞两次，并收集细胞10分钟。 </li><li>在每皿加入500μl裂解缓冲液A裂解细胞沉淀液（20mM HEPES pH值7.9，18毫米氯化钾，0.2mM的EGTA，1.5mM的氯化镁，20％甘油，0.1％NP-40），其包括蛋白酶Inhibitors鸡尾酒，1μM的TSA和在4℃下恒定旋转2mM的NAM 30分钟。 </li><li>离心机在14000 xg离心在4℃下15分钟，并转移上清液至新管。 </li><li>使用缀合，如下所述琼脂糖珠粒的抗Flag抗体进行免疫沉淀。 <ol><li>添加200-300微升偶联至琼脂糖珠的蛋白质裂解物（10-20毫克总蛋白）抗Flag抗体的。 </li><li>在4℃下以恒定的搅拌温育过夜。 </li><li>收集离心免疫沉淀在1000 xg离心在4℃下5分钟。 </li><li>洗涤沉淀2次，用10ml裂解缓冲液A以1000 xg离心在4℃下5分钟。每次洗涤后，沉淀珠子和替换用裂解缓冲液A中的上清</li><li>洗涤沉淀，用10ml裂解缓冲液A 3次无NAM在1000×g离心5分钟，4℃。每次洗涤后，沉淀珠子和替换用裂解缓冲液A.上清液它性重要NT不携带在任何不结盟运动的脱乙酰反应。 </li></ol></li><li>制备1×FLAG肽溶液（包括蛋白酶抑制剂和1μM的TSA在PBS中）。 </li><li>加入500微升1×FLAG肽溶液至琼脂糖珠孵育下恒定旋转，在4℃，30分钟。 </li><li>收集离心上清在6000 xg离心在4℃下2分钟。 </li><li>重复前两个步骤。 </li><li>通过使用过滤管和离心15,000 XG 1分钟，4℃卸下上清珠。 </li><li>使用超滤膜，浓缩洗脱样品，直至蛋白质浓度为1微克/微升。 </li><li>通过在4-12％SDS-PAGE凝胶上运行1微升洗脱的样品进行电泳。 </li><li> （:1000稀释1）和蛋白质底物（稀释取决于所用的特异性抗体）使用针对交流-K抗体western印迹确认蛋白 - 底物的乙酰化。 </li><li>保持普里田间在-80℃下的乙酰化蛋白 - 底物。 </li></ol></li><li> 体外脱乙酰反应<ol><li>制备脱乙酰缓冲液B（50mM的Tris-盐酸，pH为7.5，150 mM氯化钠，1mM的MgCl 2的0.5微米，TSA） </li><li>准备在不同管3不同的反应在20μl的最终体积（ 见表1）。 </li><li>包括额外的反应由SIRT2（可选）确认蛋白底物脱乙酰化。添加纯化的脱乙酰无效突变SIRT2 2微克到反应，而不是脱乙酰活性SIRT2（这可以使用在1.1中描述的协议来完成使用PCDH-普罗-GFP-SIRT2 H187Y -flag慢病毒后），或添加的10mM烟酰胺（NAM ）反应以抑制SIRT2的脱乙酰酶活性（ 表1）。 </li><li>孵育反应为在30℃下持续搅拌3小时。 </li><li>通过加入20微升2×样品缓冲液终止反应，煮沸样品F或在95℃下10分钟。 </li><li>通过运行在4-12％SDS-PAGE凝胶的反应混合物进行电泳和使用抗AC-κ ​​抗体（1:1000稀释）检测由印迹蛋白质-底物的乙酰化状态19,20。 </li></ol></li></ol> 2. 在体内脱乙酰含量<ol><li> SIRT2表达在培养的细胞<ol><li>准备与HEK 293T细胞10厘米培养皿稳定过表达PCDH-普罗-GFP-空载体，PCDH-普罗-GFP-SIRT2-旗及PCDH-普罗-GFP-SIRT2 H187Y -flag（如在1.1中描述）为约70 ％汇合。 </li><li>另外，准备10厘米培养皿与细胞约70％汇合，并使用上述在3微升PEI /微克DNA比使用PEI提到的载体的5微克执行瞬时表达。 </li><li>执行使用抗Flag抗体总细胞提取物的蛋白质印迹（1:1000稀释）来演示的标示 - 叔过表达agged SIRT2 19。 </li></ol></li><li> RNA干扰拦截SIRT2在培养的细胞<ol><li>制备的HEK 293T细胞的10cm的培养皿为约70％汇合的第二天。 </li><li>第二天，共转染4微克pLKO1-普罗-SH SIRT2（慢病毒载体）19，4微克pCMV- dR8.2 DVPR（包装载体）和0.5微克的pCMV-VSV-G（包络线矢量）18使用聚乙烯亚胺（PEI）在3微升的PEI /微克DNA的一比值。 注意:对于撞倒SIRT2，我们用简单的发夹的shRNA由RNAi协会（TRC）设计了pLKO.1慢病毒载体。 </li><li>操作与1.1中描述的生产shSIRT2慢病毒并感染靶细胞拦截SIRT2。 </li><li> （:1000稀释1）在10％SDS-PAGE凝胶上运行总蛋白的40微克后表现出有效的SIRT2敲低使用抗SIRT2抗体进行总细胞提取物的Western印迹。 </li><li>此外，与细胞约70％汇合准备10厘米培养皿和使用以下制造商的说明的siRNA进行SIRT2短暂击倒。 </li><li> （:1000稀释1）在10％SDS-PAGE凝胶上运行总蛋白的40微克后表现出有效的SIRT2敲低使用抗SIRT2抗体进行总细胞提取物的Western印迹。 </li></ol></li><li>免疫沉淀和免疫印迹检测培养细胞乙酰化水平<ol><li>从任一细胞过度SIRT2收集细胞沉淀之前12小时（见2.1以上）或SIRT2撞倒的细胞（见上文2.2），添加1微米的TSA到培养基中，以抑制非III类组蛋白脱乙酰。 </li><li>除去培养基，在4℃下以1000 xg离心在PBS洗涤细胞两次，并收集通过胰蛋白酶的细胞，然后离心10分钟。 </li><li>加入10 mL裂解液A（20毫米的HEPES pH值7.9，18毫米氯化钾，0.2毫米EGTA，1.5毫MgCl 2的，20％甘油，0.1％NP-40），其包括蛋白酶抑制剂鸡尾酒，1μM的TSA和2mM NAM。 </li><li>孵育在4℃下恒定旋转30分钟。 </li><li>离心收集上清在20000 XG在4°C 15分钟。 </li><li>通过使用Bradford蛋白测定计算蛋白质浓度。 </li><li>转印1mg总蛋白质的新管中的使用裂解缓冲液A.使用表2作为参考1ml的最终体积，以从细胞中任一过表达SIRT2或SIRT2击倒后制备蛋白质样品。 </li><li>执行使用针对特定的蛋白质 - 底物的抗体与蛋白A / G一起免疫沉淀（建议将40μl珠浆料）。或者，使用偶联到琼脂糖珠防AC-κ抗体（20μL珠浆通常是不够的）免疫沉淀所有的蛋白质乙酰化。 </li><li>在根据CONSTA 4℃孵育样本NT旋转过夜。 </li><li>收集离心免疫沉淀在1000 xg离心在4℃下2分钟。 </li><li>在4℃下洗珠5次用1毫升缓冲液A以1000 xg离心2分钟。 </li><li>珠悬浮在40μl的2X样品缓冲液。 </li><li>煮沸样品在95℃下10分钟。 </li><li>通过运行在4-12％SDS-PAGE凝胶洗脱的样品（从步骤2.3.12 40微升），而不会干扰珠粒在传送洗脱的样品进行电泳。 </li><li>使用抗AC-κ抗体通过Western印迹法检测蛋白质 - 底物的乙酰化水平（1:1,000稀释度），如果是用于免疫沉淀的蛋白质 - 底物特异性抗体，或针对该蛋白 - 底物的特异性抗体（遵循建议特异性抗体对稀释）如果被用于免疫沉淀的抗徒-K珠。 </li></ol></li></ol>

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H., Pirinen, E., Auwerx, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sirtuins+as+regulators+of+metabolism+and+healthspan.">Sirtuins as regulators of metabolism and healthspan.</a> Nature reviews. Molecular Cell Biology. 13, 225-238 (2012).</li><li>Choudhary, C., Weinert, B. T., Nishida, Y., Verdin, E., Mann, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+growing+landscape+of+lysine+acetylation+links+metabolism+and+cell+signalling.">The growing landscape of lysine acetylation links metabolism and cell signalling.</a> Nature Reviews. Molecular Cell Biology. 15, 536-550 (2014).</li><li>Stewart, S. A., 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=Lentivirus-delivered+stable+gene+silencing+by+RNAi+in+primary+cells.">Lentivirus-delivered stable gene silencing by RNAi in primary cells.</a> RNA. 9, 493-501 (2003).</li><li>Kim, H. S., 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=SIRT2+maintains+genome+integrity+and+suppresses+tumorigenesis+through+regulating+APC/C+activity.">SIRT2 maintains genome integrity and suppresses tumorigenesis through regulating APC/C activity.</a> Cancer Cell. 20, 487-499 (2011).</li><li>Zhang, H., 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=SIRT2+directs+the+replication+stress+response+through+CDK9+deacetylation.">SIRT2 directs the replication stress response through CDK9 deacetylation.</a> Proceedings of the National Academy of Sciences of the United States of America. 110, 13546-13551 (2013).</li><li>Weinert, B. T., 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=Proteome-wide+mapping+of+the+Drosophila+acetylome+demonstrates+a+high+degree+of+conservation+of+lysine+acetylation.">Proteome-wide mapping of the Drosophila acetylome demonstrates a high degree of conservation of lysine acetylation.</a> Science Signaling. 4, ra48 (2011).</li><li>Still, A. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantification+of+mitochondrial+acetylation+dynamics+highlights+prominent+sites+of+metabolic+regulation.">Quantification of mitochondrial acetylation dynamics highlights prominent sites of metabolic regulation.</a> The Journal of Biological Chemistry. 288, 26209-26219 (2013).</li><li>Sadoul, K., Wang, J., Diagouraga, B., Khochbin, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+tale+of+protein+lysine+acetylation+in+the+cytoplasm.">The tale of protein lysine acetylation in the cytoplasm.</a> Journal of Biomedicine &amp; Biotechnology. 2011, 970382 (2011).</li><li>Preble, J. 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=Rapid+isolation+and+purification+of+mitochondria+for+transplantation+by+tissue+dissociation+and+differential+filtration.">Rapid isolation and purification of mitochondria for transplantation by tissue dissociation and differential filtration.</a> Journal of Visualized Experiments : JoVE. , e51682 (2014).</li><li>Garcia-Cazarin, M. L., Snider, N. N., Andrade, F. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+isolation+from+skeletal+muscle.">Mitochondrial isolation from skeletal muscle.</a> Journal of Visualized Experiments : JoVE. , (2011).</li><li>He, W., Newman, J. C., Wang, M. Z., Ho, L., Verdin, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+sirtuins:+regulators+of+protein+acylation+and+metabolism.">Mitochondrial sirtuins: regulators of protein acylation and metabolism.</a> Trends in Endocrinology and Metabolism: TEM. 23, 467-476 (2012).</li></ol>

作者什么都没有透露。

Recent high throughput proteomic studies have established acetylation as a widespread PTM found not only in nucleus but also in cytoplasm and mitochondria 7,8,21-23. Taking into account the likelihood that many more acetylated proteins and sites might have been not detected due to several reasons, such as specificity of the anti-Ac-K antibodies used, the low abundance of the acetylated proteins, and the transient nature of the PTM, it is safe to predict that more acetylated proteins remain to be discovered in ...

Post-translational modifications (PTMs) regulate cell signaling networks allowing cells to rapidly respond to internal and external signals. Over the last few decades, many different PTMs playing a pivotal role in diverse processes have been identified but only a few have been studied extensively, such as phosphorylation, acetylation and ubiquitination 1-3. Focusing on acetylation, Allfrey et al. were the first to propose a role for histone acetylation in regulating gene transcription about 50 years ago 4. Research in this field has revealed that histone lysine acetylation modulates chromatin condensation and it is considered to be an epigenetic mark as part of the histone code 5. Although it took a long time until the discovery of tubulin as the first non-histone acetylation target 6, it is well established now that hundreds of eukaryotic proteins beyond histones can be acetylated and lysine acetylation has been recognized as a wide-spread PTM that may rival phosphorylation and ubiquitination in its prevalence 7-9. Interestingly, non-histone acetylated proteins can be signaling molecules in the cytoplasm, transcription factors in the nucleus, and metabolic enzymes in mitochondria, highlighting the significance of acetylation in regulating a plethora of cellular processes. 
The acetylation status of a protein depends on the coordinated and opposing function of lysine acetyltransferases (KATs) and lysine deacetylases (KDACs) which add and remove acetyl groups from proteins. The reversible acetylation of lysine, which involves neutralization of a positive charge 10, alters protein structure and it seems very likely to also alter enzymatic function in several cases 11-13. Focusing on KDACs, 18 proteins have been identified in the human and mouse genomes 14-16. Among them, mammalian sirtuins (also called class III histone lysine deacetylases) which are distinct from other members as they require NAD+ for their enzymatic function, have attracted extensive interest in this research field 16. In mammals, seven sirtuins (SIRT1-7) have been identified, each of them sharing a conserved 275-amino-acid catalytic core domain, which are mainly categorized according to their subcellular localization to the nucleus (SIRT1, 6, and 7), mitochondria (SIRT3, 4, and 5), or cytoplasm (SIRT2). SIRT1-3 have a robust deacetylation activity, while SIRT4 is reported to display ADP-ribosyltransferase activity, SIRT5 may function as a protein desuccinylase and demalonylase, and SIRT6 and SIRT7 display weak deacetylase activity but are involved in other types of acylations 17. In accordance with the significance of acetylation as a regulatory PTM modification involved in several cellular functions, sirtuins have also been implicated in a wide range of processes. After the first breakthrough studies establishing the role of sirtuins in life span extension, it has been shown that they are involved in diverse cellular functions including DNA repair, maintenance of genomic instability, apoptosis, response to stress and inflammation, control of energy efficiency, circadian clocks and metabolism, as well as contributing to the initiation and/or progression of age-related diseases such as cancer, neurodegeneration and type 2 diabetes 15,16.
Despite the significant progress in the field of sirtuin biology, more work remains to unravel undiscovered roles and functions through the identification of novel substrates. This is evidenced more emphatically by recent advances in high-resolution mass spectrometry (MS) based proteomics which have significantly increased the number of proteins found to be acetylated but most importantly have identified several different acetylated lysines in each protein, arguing that acetylation may be as wide-spread as other PTMs such as phosphorylation 7,8,17. Taking into consideration that specific deacetylases have not yet been identified for most of these acetylated proteins-substrates, it is reasonable to suggest that both in vitro and in vivo deacetylation assays are needed to confirm and establish an acetylated protein as a legitimate substrate of a specific deacetylase. In the experimental protocols described below, details will be given on how to perform both in vitro and in vivo deacetylation assays using SIRT2 as the specific deacetylase.

<table border="0" cellpadding="0" cellspacing="0" width="1543">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:459px;">cell culture dishes</td>
			<td style="width:239px;">Denville Scientific Inc.</td>
			<td style="width:119px;">T1110 and T1115</td>
			<td style="width:728px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:459px;">pCDH-puro-GFP lentiviral vector</td>
			<td style="width:239px;">System Biosciences</td>
			<td style="width:119px;">CD513B-1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">pCMV- dR8.2 dvpr (packaging vector)</td>
			<td style="width:239px;">Addgene</td>
			<td>8455</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">pCMV-VSV-G (envelope vector)</td>
			<td style="width:239px;">Addgene</td>
			<td align="right">8454</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">polyethylenimine (PEI)&#160;</td>
			<td>Polysciences Inc.</td>
			<td align="right" style="width:119px;">24885</td>
			<td>other transfection reagents can be used as well. PEI is cost effective and very efficient in transfecting 293T cells</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">0.22&#956;m filters</td>
			<td style="width:239px;">Denville Scientific Inc.</td>
			<td style="width:119px;">F5512</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">polybrene</td>
			<td style="width:239px;">Sigma</td>
			<td style="width:119px;">H9268</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;">fluorescent microscope</td>
			<td style="width:239px;">Carl Zeiss MicroImaging Inc.</td>
			<td style="width:119px;">Axiovert 200</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">puromycin</td>
			<td style="width:239px;">Invivogen</td>
			<td style="width:119px;">A11138-03</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PBS</td>
			<td style="width:239px;">Corning</td>
			<td style="width:119px;">21-031-CM</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">anti-Flag antibody</td>
			<td style="width:239px;">Sigma</td>
			<td>F3165</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">HEPES&#160;</td>
			<td style="width:239px;">Sigma</td>
			<td style="width:119px;">H3375</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">KCl</td>
			<td style="width:239px;">Sigma</td>
			<td style="width:119px;">P9541</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Glycerol</td>
			<td style="width:239px;">Sigma</td>
			<td style="width:119px;">G5516</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NP-40</td>
			<td style="width:239px;">Sigma</td>
			<td style="width:119px;">74385</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MgCl2</td>
			<td style="width:239px;">Sigma</td>
			<td style="width:119px;">M9272</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:459px;">EGTA</td>
			<td style="width:239px;">Sigma</td>
			<td style="width:119px;">34596</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">protease inhibitors coctail 100x</td>
			<td style="width:239px;">Biotool</td>
			<td style="width:119px;">B14001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Trichostatin A (TSA)</td>
			<td style="width:239px;">Sigma</td>
			<td style="width:119px;">T8552</td>
			<td>selective inhibitor of class I and II histone deacetylases (HDACs) but not class II HDACs (sirtuins)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">anti-Flag agarose beads</td>
			<td style="width:239px;">Sigma</td>
			<td style="width:119px;">A2220</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">centrifuge</td>
			<td style="width:239px;">Eppendorf</td>
			<td style="width:119px;">5417R</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">rotator</td>
			<td style="width:239px;">Thermo Scientific</td>
			<td style="width:119px;">415220Q</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">filter tubes</td>
			<td style="width:239px;">Millipore</td>
			<td>UFC30HV00</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Flag peptide&#160;</td>
			<td style="width:239px;">Sigma</td>
			<td>F3290</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Vivaspin Centrifugal Concentrator</td>
			<td>Sartorius Stedim Biotech S.A.</td>
			<td>VS0102</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">SimplyBlue SafeStain solution&#160;</td>
			<td style="width:239px;">Invitrogen</td>
			<td style="width:119px;">LC6060</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NuPAGE LDS sample buffer (4x)</td>
			<td style="width:239px;">Life Techologies</td>
			<td style="width:119px;">NP0007</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">anti-Ac-K antibody</td>
			<td style="width:239px;">Cell Signaling</td>
			<td style="width:119px;">9441</td>
			<td>several anti-Ac-K antibodies are available. In our hands, the Cell Signaling antibody exhibits the highest sensitivity</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tris-HCl pH 7.5</td>
			<td style="width:239px;">Sigma</td>
			<td style="width:119px;">T5941</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">NAD+</td>
			<td style="width:239px;">Sigma</td>
			<td style="width:119px;">N0632</td>
			<td>required cofactor for sirtuins</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">nicotinamide (NAM)</td>
			<td style="width:239px;">Sigma</td>
			<td style="width:119px;">72340</td>
			<td>selective inhibitor class II HDACs (sirtuins)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">pLKO.1 lentiviral vector&#160;</td>
			<td style="width:239px;">Addgene</td>
			<td style="width:119px;">8453</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">SIRT2 si RNA&#160;</td>
			<td style="width:239px;">Qiagen</td>
			<td style="width:119px;">GS22933</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">anti-SIRT2 antibody</td>
			<td style="width:239px;">Proteintech</td>
			<td style="width:119px;">15345-1-AP</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Bradford protein assay</td>
			<td style="width:239px;">BIO-RAD</td>
			<td style="width:119px;">500-0006</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">anti Ac-K agarose beads</td>
			<td style="width:239px;">Immunechem</td>
			<td>ICP0388</td>
			<td></td>
		</tr>
	</tbody>
</table>

deacetylation assays to unravel the interplay between sirtuins (sirt2) and specific protein-substrates

Acetylation has emerged as an important post-translational modification (PTM) regulating a plethora of cellular processes and functions. This is further supported by recent findings in high-resolution mass spectrometry based proteomics showing that many new proteins and sites within these proteins can be acetylated. However the identity of the enzymes regulating these proteins and sites is often unknown. Among these enzymes, sirtuins, which belong to the class III histone lysine deacetylases, have attracted great interest as enzymes regulating the acetylome under different physiological or pathophysiological conditions. Here we describe methods to link SIRT2, the cytoplasmic sirtuin, with its substrates including both in vitro and in vivo deacetylation assays. These assays can be applied in studies focused on other members of the sirtuin family to unravel the specific role of sirtuins and are necessary in order to establish the regulatory interplay of specific deacetylases with their substrates as a first step to better understand the role of protein acetylation. Furthermore, such assays can be used to distinguish functional acetylation sites on a protein from what may be non-regulatory acetylated lysines, as well as to examine the interplay between a deacetylase and its substrate in a physiological context.

为了使蛋白质被认为是用于与脱乙酰酶活性的任何酶合法脱乙酰目标，无论是在体外和体内脱乙酰测定需要执行以建立脱乙酰酶和其底物之间的相互作用。 用于体外脱乙酰酶测定中，可以进行该测定之前，需要的脱乙酰酶和乙酰化蛋白底物两者的纯化。在这里，我们使用作为要研究的特定脱乙酰酶细胞质沉默调节蛋白SIRT2。 SIRT2是强烈建议作为?...

Watch this Scientific Journal Video about Deacetylation Assays to Unravel the Interplay between Sirtuins SIRT2 and Specific Protein-substrates at JoVE.com

Deacetylation Assays to Unravel the Interplay between Sirtuins SIRT2 and Specific Protein-substrates

This protocol describes the required steps to execute in vitro and in vivo deacetylation assays in order to establish the role of proteins as specific deacetylation substrates for sirtuins and further study the role of reversible - lysine acetylation as a post-translational modification.

脱乙酰测定解开蛋白特异底物乙酰化酶之间的Interplay（SIRT2）和

Acetylation has emerged as an important post-translational modification (PTM) regulating a plethora of cellular processes and functions. This is further ...

deacetylation-assays-to-unravel-interplay-between-sirtuins-sirt2

Research

JoVE Journal

Chemistry

Deacetylation Assays to Unravel the Interplay between Sirtuins (SIRT2) and Specific Protein-substrates

8.2K 次观看.  Feinberg School of Medicine, Northwestern University. This protocol describes the required steps to execute in vitro and in vivo deacetylation assays in order to establish the role of proteins as specific deacetylation substrates for sirtuins and further study the role of reversible - lysine acetylation as a post-translational modification.

视频: Deacetylation Assays to Unravel the Interplay between Sirtuins SIRT2 and Specific Protein-substrates

Origami Inspired Self-assembly of Patterned and Reconfigurable Particles

There are numerous techniques such as photolithography, electron-beam lithography and soft-lithography that can be used to precisely pattern two dimensional (2D) structures. These technologies are mature, offer high precision and many of them can be implemented in a high-throughput manner. We leverage the advantages of planar lithography and combine them with self-folding methods1-20 wherein physical forces derived from surface tension or residual stress, are used to curve or fold planar structures into three dimensional (3D) structures. In doing so, we make it possible to mass produce precisely patterned static and reconfigurable particles that are challenging to synthesize.
In this paper, we detail visualized experimental protocols to create patterned particles, notably, (a) permanently bonded, hollow, polyhedra that self-assemble and self-seal due to the minimization of surface energy of liquefied hinges21-23 and (b) grippers that self-fold due to residual stress powered hinges24,25. The specific protocol described can be used to create particles with overall sizes ranging from the micrometer to the centimeter length scales. Further, arbitrary patterns can be defined on the surfaces of the particles of importance in colloidal science, electronics, optics and medicine. More generally, the concept of self-assembling mechanically rigid particles with self-sealing hinges is applicable, with some process modifications, to the creation of particles at even smaller, 100 nm length scales22, 26 and with a range of materials including metals21, semiconductors9 and polymers27. With respect to residual stress powered actuation of reconfigurable grasping devices, our specific protocol utilizes chromium hinges of relevance to devices with sizes ranging from 100 &mu;m to 2.5 mm. However, more generally, the concept of such tether-free residual stress powered actuation can be used with alternate high-stress materials such as heteroepitaxially deposited semiconductor films5,7 to possibly create even smaller nanoscale grasping devices.

We describe experimental details of the synthesis of patterned and reconfigurable particles from two dimensional (2D) precursors. This methodology can be used to create particles in a variety of shapes including polyhedra and grasping devices at length scales ranging from the micro to centimeter scale.

折纸灵感的图案和可重构颗粒自组装

There are numerous techniques such as photolithography, electron-beam lithography and soft-lithography that can be used to precisely pattern two ...

科研

化学

Rapid Colorimetric Assays to Qualitatively Distinguish RNA and DNA in Biomolecular Samples

Biochemical experimentation generally requires accurate knowledge, at an early stage, of the nucleic acid, protein, and other biomolecular components in potentially heterogeneous specimens. Nucleic acids can be detected via several established approaches, including analytical methods that are spectrophotometric (e.g., A260), fluorometric (e.g., binding of fluorescent dyes), or colorimetric (nucleoside-specific chromogenic chemical reactions).1 Though it cannot readily distinguish RNA from DNA, the A260/A280 ratio is commonly employed, as it offers a simple and rapid2 assessment of the relative content of nucleic acid, which absorbs predominantly near 260 nm and protein, which absorbs primarily near 280 nm. Ratios &lt; 0.8 are taken as indicative of 'pure' protein specimens, while pure nucleic acid (NA) is characterized by ratios &gt; 1.53.
However, there are scenarios in which the protein/NA content cannot be as clearly or reliably inferred from simple uv-vis spectrophotometric measurements. For instance, (i) samples may contain one or more proteins which are relatively devoid of the aromatic amino acids responsible for absorption at &asymp;280 nm (Trp, Tyr, Phe), as is the case with some small RNA-binding proteins, and (ii) samples can exhibit intermediate A260/A280 ratios (~0.8 &lt; ~1.5), where the protein/NA content is far less clear and may even reflect some high-affinity association between the protein and NA components. For such scenarios, we describe herein a suite of colorimetric assays to rapidly distinguish RNA, DNA, and reducing sugars in a potentially mixed sample of biomolecules. The methods rely on the differential sensitivity of pentoses and other carbohydrates to Benedict's, Bial's (orcinol), and Dische's (diphenylamine) reagents; the streamlined protocols can be completed in a matter of minutes, without any additional steps of having to isolate the components. The assays can be performed in parallel to differentiate between RNA and DNA, as well as indicate the presence of free reducing sugars such as glucose, fructose, and ribose (Figure 1).

A suite of colorimetric assays is described for rapidly distinguishing protein, RNA, DNA, and reducing sugars in potentially heterogeneous biomolecular samples.

快速比色测定RNA和DNA生物分子样品进行定性区分

Biochemical experimentation generally requires accurate knowledge, at an early stage, of the nucleic acid, protein, and other biomolecular components in ...

Isolation and Chemical Characterization of Lipid A from Gram-negative Bacteria

Lipopolysaccharide (LPS) is the major cell surface molecule of gram-negative bacteria, deposited on the outer leaflet of the outer membrane bilayer. LPS can be subdivided into three domains: the distal O-polysaccharide, a core oligosaccharide, and the lipid A domain consisting of a lipid A molecular species and 3-deoxy-D-manno-oct-2-ulosonic acid residues (Kdo). The lipid A domain is the only component essential for bacterial cell survival. Following its synthesis, lipid A is chemically modified in response to environmental stresses such as pH or temperature, to promote resistance to antibiotic compounds, and to evade recognition by mediators of the host innate immune response. The following protocol details the small- and large-scale isolation of lipid A from gram-negative bacteria. Isolated material is then chemically characterized by thin layer chromatography (TLC) or mass-spectrometry (MS). In addition to matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) MS, we also describe tandem MS protocols for analyzing lipid A molecular species using electrospray ionization (ESI) coupled to collision induced dissociation (CID) and newly employed ultraviolet photodissociation (UVPD) methods. Our MS protocols allow for unequivocal determination of chemical structure, paramount to characterization of lipid A molecules that contain unique or novel chemical modifications. We also describe the radioisotopic labeling, and subsequent isolation, of lipid A from bacterial cells for analysis by TLC. Relative to MS-based protocols, TLC provides a more economical and rapid characterization method, but cannot be used to unambiguously assign lipid A chemical structures without the use of standards of known chemical structure. Over the last two decades isolation and characterization of lipid A has led to numerous exciting discoveries that have improved our understanding of the physiology of gram-negative bacteria, mechanisms of antibiotic resistance, the human innate immune response, and have provided many new targets in the development of antibacterial compounds.

Isolation and characterization of the lipid A domain of lipopolysaccharide (LPS) from gram-negative bacteria provides insight into cell surface based mechanisms of antibiotic resistance, bacterial survival and fitness, and how chemically diverse lipid A molecular species differentially modulate host innate immune responses.

隔离和脂质A的化学表征革兰氏阴性菌

Lipopolysaccharide (LPS) is the major cell surface molecule of gram-negative bacteria, deposited on the outer leaflet of the outer membrane bilayer. LPS ...

Structure and Coordination Determination of Peptide-metal Complexes Using 1D and 2D 1H NMR

Copper (I) binding by metallochaperone transport proteins prevents copper oxidation and release of the toxic ions that may participate in harmful redox reactions. The Cu (I) complex of the peptide model of a Cu (I) binding metallochaperone protein, which includes the sequence MTCSGCSRPG (underlined is conserved), was determined in solution under inert conditions by NMR spectroscopy.
NMR is a widely accepted technique for the determination of solution structures of proteins and peptides. Due to difficulty in crystallization to provide single crystals suitable for X-ray crystallography, the NMR technique is extremely valuable, especially as it provides information on the solution state rather than the solid state. Herein we describe all steps that are required for full three-dimensional structure determinations by NMR. The protocol includes sample preparation in an NMR tube, 1D and 2D data collection and processing, peak assignment and integration, molecular mechanics calculations, and structure analysis. Importantly, the analysis was first conducted without any preset metal-ligand bonds, to assure a reliable structure determination in an unbiased manner.

Structure and Coordination Determination of Peptide-metal Complexes Using 1D and 2D 1H NMR

The NMR-solution structure of a metallochaperone model peptide with Cu (I) was determined, and a detailed protocol from sample preparation and 1D and 2D data collection to a three-dimensional structure is described.

Copper (I) binding by metallochaperone transport proteins prevents copper oxidation and release of the toxic ions that may participate in harmful redox ...

Split-and-pool Synthesis and Characterization of Peptide Tertiary Amide Library

Peptidomimetics are great sources of protein ligands. The oligomeric nature of these compounds enables us to access large synthetic libraries on solid phase by using combinatorial chemistry. One of the most well studied classes of peptidomimetics is peptoids. Peptoids are easy to synthesize and have been shown to be proteolysis-resistant and cell-permeable. Over the past decade, many useful protein ligands have been identified through screening of peptoid libraries. However, most of the ligands identified from peptoid libraries do not display high affinity, with rare exceptions. This may be due, in part, to the lack of chiral centers and conformational constraints in peptoid molecules. Recently, we described a new synthetic route to access peptide tertiary amides (PTAs). PTAs are a superfamily of peptidomimetics that include but are not limited to peptides, peptoids and N-methylated peptides. With side chains on both &#945;-carbon and main chain nitrogen atoms, the conformation of these molecules are greatly constrained by sterical hindrance and allylic 1,3 strain. (Figure 1) Our study suggests that these PTA molecules are highly structured in solution and can be used to identify protein ligands. We believe that these molecules can be a future source of high-affinity protein ligands. Here we describe the synthetic method combining the power of both split-and-pool and sub-monomer strategies to synthesize a sample one-bead one-compound (OBOC) library of PTAs.

Peptide tertiary amides (PTAs) are a superfamily of peptidomimetics that include but are not limited to peptides, peptoids and N-methylated peptides. Here we describe a synthetic method which combines&#160;both split-and-pool and sub-monomer strategies to synthesize a one-bead one-compound library of PTAs.

分裂和池合成及肽叔酰胺图书馆表征

Peptidomimetics are great sources of protein ligands. The oligomeric nature of these compounds enables us to access large synthetic libraries on solid ...

Amide Coupling Reaction for the Synthesis of Bispyridine-based Ligands and Their Complexation to Platinum as Dinuclear Anticancer Agents

Amide coupling reactions can be used to synthesize bispyridine-based ligands for use as bridging linkers in multinuclear platinum anticancer drugs. Isonicotinic acid, or its derivatives, are coupled to variable length diaminoalkane chains under an inert atmosphere in anhydrous DMF or DMSO with the use of a weak base, triethylamine, and a coupling agent, 1-propylphosphonic anhydride. The products precipitate from solution upon formation or can be precipitated by the addition of water. If desired, the ligands can be further purified by recrystallization from hot water. Dinuclear platinum complex synthesis using the bispyridine ligands is done in hot water using transplatin. The most informative of the chemical characterization techniques to determine the structure and gross purity of both the bispyridine ligands and the final platinum complexes is 1H NMR with particular analysis of the aromatic region of the spectra (7-9 ppm). The platinum complexes have potential application as anticancer agents and the synthesis method can be modified to produce trinuclear and other multinuclear complexes with different hydrogen bonding functionality in the bridging ligand.

This protocol describes the use of amide coupling reactions of isonicotinic acid and diaminoalkanes to form bridging ligands suitable for use in the synthesis of multinuclear platinum complexes, which combine aspects of the anticancer drugs BBR3464 and picoplatin.

酰胺偶联反应为双吡啶基配体及其络合合成，以白金为双核抗癌药物

Amide coupling reactions can be used to synthesize bispyridine-based ligands for use as bridging linkers in multinuclear platinum anticancer drugs. ...

Characterization, Quantification and Compound-specific Isotopic Analysis of Pyrogenic Carbon Using Benzene Polycarboxylic Acids (BPCA)

Fire-derived, pyrogenic carbon (PyC), sometimes called black carbon (BC), is the carbonaceous solid residue of biomass and fossil fuel combustion, such as char and soot. PyC is ubiquitous in the environment due to its long persistence, and its abundance might even increase with the projected increase in global wildfire activity and the continued burning of fossil fuel. PyC is also increasingly produced from the industrial pyrolysis of organic wastes, which yields charred soil amendments (biochar). Moreover, the emergence of nanotechnology may also result in the release of PyC-like compounds to the environment. It is thus a high priority to reliably detect, characterize and quantify these charred materials in order to investigate their environmental properties and to understand their role in the carbon cycle.
Here, we present the benzene polycarboxylic acid (BPCA) method, which allows the simultaneous assessment of PyC&#39;s characteristics, quantity and isotopic composition (13C and 14C) on a molecular level. The method is applicable to a very wide range of environmental sample materials and detects PyC over a broad range of the combustion continuum, i.e., it is sensitive to slightly charred biomass as well as high temperature chars and soot. The BPCA protocol presented here is simple to employ, highly reproducible, as well as easily extendable and modifiable to specific requirements. It thus provides a versatile tool for the investigation of PyC in various disciplines, ranging from archeology and environmental forensics to biochar and carbon cycling research.

Characterization, Quantification and Compound-specific Isotopic Analysis of Pyrogenic Carbon Using Benzene Polycarboxylic Acids BPCA

We present the benzene polycarboxylic acid (BPCA) method for assessing pyrogenic carbon (PyC) in the environment. The compound-specific approach uniquely provides simultaneous information about the characteristics, quantity and isotopic composition (13C and 14C) of PyC.

表征，气相炭用苯聚羧酸的定量和特定化合物同位素分析（BPCA）

Fire-derived, pyrogenic carbon (PyC), sometimes called black carbon (BC), is the carbonaceous solid residue of biomass and fossil fuel combustion, such as ...

Using Capillary Electrophoresis to Quantify Organic Acids from Plant Tissue: A Test Case Examining Coffea arabica Seeds

Carboxylic acids are organic acids containing one or more terminal carboxyl (COOH) functional groups. Short chain carboxylic acids (SCCAs; carboxylic acids containing three to six carbons), such as malate and citrate, are critical to the proper functioning of many biological systems, where they function in cellular respiration and can serve as indicators of cell health. In foods, organic acid content can have significant impact on taste, with increased SCCA levels resulting in a sour or &#34;acid&#34; taste. Because of this, methods for the rapid analysis of organic acid levels are of particular interest to the food and beverage industries. Unfortunately, however, most methods used for SCCA quantification are dependent on time-consuming protocols requiring the derivatization of samples with hazardous reagents, followed by costly chromatographic and/or mass spectrometric analyses. This method details an alternate method for the detection and quantification of organic acids from plant material and food samples using free zonal capillary electrophoresis (CZE), sometimes simply referred to as capillary electrophoresis (CE). CZE provides a cost-effective method for measuring SCCAs with a low limit of detection (0.005 mg/ml). This article details the extraction and quantification of SCCAs from plant samples. While the method provided focuses on measurement of SCCAs from coffee beans, the method provided can be applied to multiple plant-based food materials.

Using Capillary Electrophoresis to Quantify Organic Acids from Plant Tissue: A Test Case Examining Coffea arabica Seeds

This article presents a method for the detection and quantification of organic acids from plant material using free zonal capillary electrophoresis. An example of the potential application of this method, determining the effects of a secondary fermentation on organic acid levels in coffee seeds, is provided.

利用毛细管电泳从植物组织量化有机酸:一个测试用例检查<em&gt;小粒种咖啡</em&gt;种子

Carboxylic acids are organic acids containing one or more terminal carboxyl (COOH) functional groups. Short chain carboxylic acids (SCCAs; carboxylic ...

Coulomb Explosion Imaging as a Tool to Distinguish Between Stereoisomers

This article shows how the COLTRIMS (Cold Target Recoil Ion Momentum Spectroscopy) or the "reaction microscope" technique can be used to distinguish between enantiomers (stereoisomers) of simple chiral species on the level of individual molecules. In this approach, a gaseous molecular jet of the sample expands into a vacuum chamber and intersects with femtosecond (fs) laser pulses. The high intensity of the pulses leads to fast multiple ionization, igniting a so-called Coulomb Explosion that produces several cationic (positively charged) fragments. An electrostatic field guides these cations onto time- and position-sensitive detectors. Similar to a time-of-flight mass spectrometer, the arrival time of each ion yields information on its mass. As a surplus, the electrostatic field is adjusted in a way that the emission direction and the kinetic energy after fragmentation lead to variations in the time-of-flight and in the impact position on the detector.
Each ion impact creates an electronic signal in the detector; this signal is treated by high-frequency electronics and recorded event by event by a computer. The registered data correspond to the impact times and positions. With these data, the energy and the emission direction of each fragment can be calculated. These values are related to structural properties of the molecule under investigation, i.e. to the bond lengths and relative positions of the atoms, allowing to determine molecule by molecule the handedness of simple chiral species and other isomeric features.

For small chiral species, Coulomb Explosion Imaging provides a new approach to determine the handedness of individual molecules.

作为一种工具来区分立体异构体的库仑爆炸成像

This article shows how the COLTRIMS (Cold Target Recoil Ion Momentum Spectroscopy) or the "reaction microscope" technique can be used to distinguish ...

Raman and IR Spectroelectrochemical Methods as Tools to Analyze Conjugated Organic Compounds

In the presented work, two spectroelectrochemical techniques are discussed as tools for the analysis of the structural changes occurring in the molecule on the vibrational level of energy. Raman and IR spectroelectrochemistry can be used for advanced characterization of the structural changes in the organic electroactive compounds. Here, the step-by-step analysis by means of Raman and IR spectroelectrochemistry is shown. Raman and IR spectroelectrochemical techniques provide complementary information about structural changes occurring during an electrochemical process, i.e. allows for the investigation of redox processes and their products. The examples of IR and Raman spectroelectrochemical analysis are presented, in which the products of the redox reactions, both in solution and solid state, are identified.

A protocol of step-by-step Raman and IR spectroelectrochemical analysis is presented.

拉曼光谱和红外光谱电化学法作为分析共轭有机化合物的工具

In the presented work, two spectroelectrochemical techniques are discussed as tools for the analysis of the structural changes occurring in the molecule ...