这项工作是由美国弗吉尼亚大学和Jeffress纪念信托基金（J-971）。我们感谢K. L.哥伦布，耆那教，P.兰多夫的有益讨论和批判性阅读的手稿。

1。本笃十六世为还原糖的含量<ol><li>本笃十六世的试剂准备一个合适的数量 - 940毫米无水碳酸钠，588 mM柠檬酸钠脱水，68毫米铜（II）五水硫酸铜。这种试剂可以存储在室温（RT）为至少6个月，没有明显的反应性变化。 </li><li>上述试剂是6倍。因此，600μL的反应中，加入100μl本笃十六世试剂一干净的1.5ml离心管（ 如 Eppendorf公司品牌），每个样品进行检测。 </li><li>加入任何地方从10微升至500微升的样品此管，在最初的试验运行，基于颜色形成的强度，可确定最佳的音量。如果有足够的采样，是可用的，那么开始进行这种试验在最大可能的样品的体积（ 即 ，六分之五的整体的反应体积，在这种情况下，将500μl的样品），然后在随后的测定稀释。 </li><li> DDH 2 O涂是，使终体积为600微升;混合溶液通过涡旋或移液。 </li><li>在沸水浴中20分钟，孵育样品。 </li><li>从浴中取出加热样品，并让它冷却10分钟，在RT。 </li><li>离心样品管在&gt; 9,300 xg离心（10,000 rpm下在一个FA45-24-11的Eppendorf固定角转子）为5分钟，以沉积物任何颗粒材料;此步骤中更重要的是定量而非定性研究。 </li><li>等分试样的上清液从该管到一个干净的比色皿中。 </li><li>空白，紫外可见分光光度计水。 </li><li>该样品在475 nm处测定吸光度。 </li></ol> 2。 BIAL的地衣酚含量为戊糖<ol><li>准备一个合适的数量的新鲜BIAL试剂 - 24.2 mM的地衣酚一水合物（见该化合物的结构的图2b），6 M的HCl，0.025％w / v的氯化铁六水合物。 注:扩展存储的BIAL的试剂可以制备作为两个单独的库存的解决方案:（ⅰ）试剂A [0.05％w / v的氯化铁•6H 2ò在浓HCl]（ⅱ）试剂乙[422 mM的苔黑素一水合物制备在95 ％的乙醇。试剂A可以存​​储在RT六个月;试剂B可以在4℃下存储一个月，用箔片覆盖，以限制曝光。这些原液中的图15（A）:1（B）的体积/体积比在使用前混合。 </li><li>上述试剂是2x。因此，为1.0毫升的反应，添加500μl的BIAL试剂，洁净的1.5 ml离心管中，每个样品进行检测。 </li><li>加入任何地方从10微升至500微升的样品本管。至于每个音符的1.3（上述）中，如果有足够的采样，是可用的，那么开始试使用最大可能的样品的体积（ 即 ，一个一半的总的反应体积中，在这种情况下，将500μl的样品）的反应，并从那里稀释。 </li><li>加入DDH 2 O至管带来的final量为1.0 mL，涡旋式或移液混合溶液。 </li><li>在沸水浴中20分钟，孵育样品。 </li><li>从浴中取出加热样品，并让它冷却10分钟，在RT。 </li><li>离心样品管在&gt; 9,300 xg离心（10,000 rpm下在一个FA45-24-11的Eppendorf固定角转子）为5分钟，以沉积物任何颗粒材料;此步骤中更重要的是定量而非定性研究。 </li><li>从这个管分装上清到一个干净的比色皿的目视检查。 </li><li>对于半定量分析，用空白的UV-Vis分光光度计与水，并测量在660nm处的吸光度的比色皿样品。 </li></ol> 3。 Dische的苯胺含量2&#39;-deoxypentose的糖<ol><li>准备Dische的二苯胺试剂- 60mM的二苯基胺，11 M冰醋酸酸，179 mM的硫酸，62％v / v乙醇的合适的数量。这种试剂可以预先相比提前在一个黑暗的容器内，储存在室温下或用铝箔纸覆盖，以限制光暴露。由于光灵敏度的试剂不能无限期地储存，但在实践中，它可以每天两至三个月没有明显的变化反应。 </li><li>上述试剂是2x。因此，为1.0毫升的反应，添加500μl的Dische试剂，洁净的1.5 ml离心管中，每个样品进行检测。 </li><li>加入任何地方从10微升至500微升的样品本管。至于每个音符的1.3（上述）中，如果有足够的采样，是可用的，那么开始试使用最大可能的样品的体积（ 即 ，一个一半的总的反应体积中，在这种情况下，将500μl的样品）的反应，并从那里稀释。 </li><li>加入DDH 2 O至管带来终体积为1.0毫升;，通过涡旋或移液混合溶液。 </li><li>在沸水浴中20分钟，孵育样品。 </li><li>取出加热样品的浴缸和异体瓦特它在室温冷却10分钟。 </li><li>离心样品管在&gt; 9,300 xg离心（10,000 rpm下在一个FA45-24-11的Eppendorf固定角转子）为5分钟，以沉积物任何颗粒材料;此步骤中更重要的是定量而非定性研究。 </li><li>从这个管分装上清到一个干净的比色皿的目视检查。 </li><li>对于半定量分析，用空白的UV-Vis分光光度计与水，并测量在600nm处的吸光度的比色皿样品。 </li></ol> 4。进一步的使用注意事项<ol><li>下面的类的分子化合物，每个检测为阳性和阴性对照反应是合适的参考: <ul><li>本笃十六世-正面的游离核糖，果糖，葡萄糖，负= RNA，DNA，ATP 等 。 （任何缺乏自由还原糖功能的糖类） </li><li> BIAL -正RNA（ 如面包酵母提取物），核糖，磷酸腺苷，UMP;负=牛SER这个白蛋白（BSA）或任何其他蛋白质</li><li> Dische -正= 如小牛胸腺DNA（）;负RNA，ATP 等 。 （任何非-2&#39;-脱氧核苷酸） </li></ul></li><li>这些测定法已发现常常在蛋白质纯化中使用的化合物是相当的弹性，例如，常见的盐，如NaCl，（NH 4）2 SO 4，K 2 SO 4没有干扰，而出现的反应一般不受的稀释剂的内容。洗涤剂或离液序列高的剂如尿素可能会影响测定的反应性，如果pH值是高碱性;一个中性至酸性pH值范围是一般最优化的BIAL的和Dische的检测，因为在底层反应化学（见的文本和质子在图2b中，三 ）。潜在的不理想的反应条件下，怀疑是干扰物等 。应在逐案基础测试，使用阳性和阴性对照实验瓦特第i个参考化合物。 </li></ol>

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没有利益冲突的声明。

<html>比色法这里提供一个简单的方法来快速评估，如时遇到的纯化蛋白质，RNA或全细胞裂解物的复合物，准备进一步研究生物分子混合物的化学性质。结构生物学追求更自然的组件，逐步更大的挑战，如样本的异质性，将所构成的复杂和多组分复合物。超分子组装往往只有轻微稳定，他们的成功分离纯化条件可能要求不那么严格（ 例如 ，如发现剪接snRNP复合17,18）。在这样的温和?...

许多细胞生物学发生通过涉及DNA和RNA的分子间的相互作用。4这些天然存在的核酸（NAS）彼此互动，5与蛋白质，第6和在体内与主机的小分子化合物和配位体（ 例如 ，二价阳离子7）。的相互作用可能是短期或长期的（动力学），范围从高至中度到低亲和力的（热力学实力），而且也表现在化学性质上的巨大变化和特异性-某些团体是很特别的（ 例如 ，DNA· ··转录因子，RNA···剪接因子），而其他的相互作用是必然远比更通用的（ 例如 ，DNA···细菌组蛋白样胡蛋白质8）。可以有非特异性相互作用与定居在体外实验中，涉及的混合物biomo实际后果lecules，因为它是可能的，甚至是有可能的，一些定居将与感兴趣的生物分子的关联，至少在所使用的实验条件下的一些子集（离子强度，溶液pH值， 等 ）。 考虑，例如， 通过异源表达的重组蛋白在大肠杆菌中的细胞培养生产的一种蛋白质（POI）的感兴趣的，在几乎任何结构生物学实验室，这样的程序进行例行9在进一步的实验中准备的，例如生物化学/生物物理特性，结晶， 等 ，最初的努力一般都集中于获得足够数量的POI作为尽可能纯的一种形式，理想状态为化学均质和生物物理单分散的试样。破坏的宿主细胞后，一个典型的纯化工作流程的早期阶段的目标是隔离的POI从大肠杆菌大肠杆菌的蛋白质，核酸，细胞壁碎片，和其他部件的细胞溶胞产物。但是，主机NAS合作净化的POI数理化的原因-一个非常基本的POI非特异性下拉宿主DNA / RNA的POI可能有一个通用的NA-约束力的活动（ 例如 ，上述HU）;该POI可能是一个相当具体的NA-结合蛋白，但表现出与宿主的RNA或DNA的交叉反应性，主机定居可能交互用色谱基质，从而简单地与POI共洗脱;及等等。不分青红皂白，高亲和性结合的主机来港定居的POI，可以构成一个棘手的问题，，因为NA杂质可能会影响下游实验（ 如荧光的各向异性实验的POI•RNA结合10）。或者，未预料到的POI的···NA协会也可以被看作偶然的，作为这种相互作用照亮​​的POI的核酸结合能力。无论哪种方式，是否定居是关键部件或污染物，一个必须首先量化和识别类型（DNA，RNA）的共同净化定居下游实验准备。 存在几种分析方法，用于检测和定量样品中定居。大多数可用的方法是从根本上无论是分光光度法（ 例如 ，A吸光度值260和A 260 / A 280的比率），荧光（ 例如 ，有约束力的噻唑橙或其他荧光染料NA），或色度（核苷化学反应的敏感性收率的发色团吸收的电磁波谱的UV-Vis区域），最近由De梅伊等人描述。1然而，识别类型的多核苷酸的RNA或DNA的关键步骤是超出了范围，许多这些定量的方法。在这里，我们提供了一套比色测定法，以迅速确定的蛋白质样品中的NA组件的类型。 该协议描述这里c可以有效地执行而无需额外的步骤中分离的潜在NA杂质，和依靠Benedict的测定用于减少糖11，地衣酚测定戊糖12,13，和二苯基胺的反应14,15的2&#39;-deoxypentoses（ 图1和图2） 。的笃的测试（ 图2a）利用的能力的线性，开放-链（醛）形式的醛糖糖以减少铜2 +，伴随氧化的糖的羰基，以一个羧酸酯基团和生产的Cu 2 O的作为不溶性沉淀。该反应将测试带免费还原糖，如醛糖和酮糖（转换到相应的醛糖通过 enediol中间体）阳性，但不与被锁定的共价骨干的DNA或RNA的多核苷酸的一部分成环状形式的戊糖。由于简约的半缩醛功能的要求，其他共同mpounds试验阳性在这个测定法- ，因此作为潜在的干扰物-包括α-羟基酮和短低聚糖（ 例如 ，二糖麦芽糖）。无论是BIAL地衣酚（ 图2b），是在初始破坏的多核苷酸骨干Dische的二苯胺（ 图2c）的反应， 通过脱嘌呤核苷和进一步的酸，或碱催化的水解的父核苷酸，使其呋喃-2甲醛（ 糠醛 ）衍生物，这些衍生物然后与多羟基酚，如地衣酚（BIAL）或二苯胺试剂形成彩色的缩合产物，很大程度上是未知的化学结构（Dische）。的DNA与RNA特异性Dische的检测源于一个事实，即与戊糖必须是2&#39;-脱氧以易被氧化的ω-hydroxylevulinyl醛，其中，还与二苯胺反应在酸性条件下产生一个明亮的蓝色凝析（ 图2c）。使用这里描述的精简协议，我们已经发现这些糖特定的比色反应可以区分的RNA和DNA，并也将表明的免费的还原糖，如葡萄糖，果糖，或核糖的生物分子样品中的存在。

<table border="1"><tbody><tr><td> 试剂或设备 </td><td> 供应商/公司 </td><td> 目录号 </td><td> 注释，说明 </td></tr><tr><td>无水碳酸钠</td><td> Fisher Scientific则</td><td> S263 </td><td></td></tr><tr><td>柠檬酸钠二水合物</td><td>西格玛</td><td> S-4641 </td><td></td></tr><tr><td>铜（II）五水硫酸铜</td><td> VWR </td><td> VW3312-2 </td><td></td></tr><tr><td>地衣酚一水物</td><td> Sigma-Aldrich公司</td><td> O1875 </td><td></td></tr><tr><td>浓盐酸</td><td> VWR </td><td> BDH3030 </td><td></td></tr><tr><td>六水三氯化铁</td><td>西格玛</td><td> F-2877 </td><td></td></tr><tr><td>二苯胺</td><td> Aldrich公司</td><td> 112763 </td><td></td></tr><tr><td>冰醋酸</td><td> Fisher Scientific则</td><td> A28 </td><td></td></tr><tr><td>硫酸</td><td> Sigma-Aldrich公司</td><td> 258105 </td><td></td></tr><tr><td>乙醇</td><td> Koptec </td><td> V1101 </td><td></td></tr><tr><td>核糖</td><td>西格玛</td><td> R-7500 </td><td>准备在1％W / V H 2 O </td></tr><tr><td>从面包酵母（ 酿酒酵母核糖核酸） </td><td>西格玛</td><td> R6750 </td><td>准备在H 2 O在10毫克/毫升;储存于-20°C </td></tr><tr><td>脱氧核糖核酸（钠盐），从小牛胸腺</td><td>西格玛</td><td> D1501 </td><td>准备在H 2 O在10毫克/毫升;商店在4°C </td></tr><tr><td></td><td></td><td></td><td> 试剂，设备与安全 材料中列出了聚焦llowing表中出现的顺序，他们的协议部分。除非另有说明，否则，（上述），可以存储所有的试剂在室温和照明。对于任何项目未在下面列出（ 例如 ，微量离心管），一般的品牌/型号/一个标准的生化实验室中发现的各种可以使用（ 例如 ，Eppendorf公司品牌的1.5 ml离心管）。标准的塑料微量离心管中，应使用涉及离心分离（ 例如 ，每个协议结束附近的沉积物的颗粒材料）的步骤。没有特别的材料是优选的，只要该管可以被密封;生物化学实验室发现的典型的聚丙烯管中工作良好。在安全的关注和废物处理方面，标准实验室的预防措施（安全眼镜，通风柜）应行使前比较，工作，和处理的解决方案，浓醋酸，盐酸或硫酸。对于有机试剂如地衣酚或DIPH烯基胺，腈手套优选共同胶乳（天然橡胶）多种。 </td></tr></tbody></table>

rapid colorimetric assays to qualitatively distinguish rna and dna in biomolecular samples

生化实验通常需要准确的知识，在早期阶段，所述的核酸，蛋白质，和其他生物分子的组成部分，在潜在的异构试样。核酸可以被检测到，通过几个既定的方法，包括分析方法，有分光光度法（ 例如 ，A 260），荧光（ 例如 ，荧光染料的结合），或比色（核苷，具体的显色化学反应的）。1虽然它可以不容易区分RNA从DNA，A 260 / A 280比值是常用的，因为它提供了一个简单，快速的评估 ，核酸的相对含量，主要吸收近260 nm和蛋白质，主要吸收280纳米附近的。视为指示"纯粹的"蛋白标本比率&lt;0.8，而纯化的核酸，其特征在于，（NA）的比率&gt; 1.5 3。 何wever，有场景，其中的蛋白质/ NA含量不能明确或可靠的推断，从简单的紫外 - 可见分光光度法测量。例如，（ⅰ）样品可能包含一个或多个蛋白质，这是相对缺乏负责吸收≈280nm处（色氨酸，酪氨酸，苯丙氨酸）的芳族氨基酸，是与一些小的RNA结合蛋白的情况下，并（二）样品可以表现出中间A 260 / A 280比值（0.8〜1.5），其中的蛋白质/ NA含量远远不太清楚，甚至可能反映了一些高亲和力的蛋白和NA之间的关联。在这样的情况下，我们描述了一套比色法快速区分RNA，DNA和还原糖的一个潜在的混合样品的生物分子。该方法依赖本笃十六世戊糖和其他碳水化合物不同的敏感度，BIAL（地衣酚），Dische（二苯胺）试剂;流线型的协议可以是COM处理完成，在短短的几分钟内，没有任何额外的步骤隔离组件。该试验可以被并行地执行，来区分的RNA和DNA，以及指示免费的还原糖，如葡萄糖，果糖，核糖（ 图1）的存在下。

结果示于图3，用于应用这些已知的参考化合物的比色测 ​​定法。代表性的定性数据笃的:（a），BIAL的地衣酚（b）中和Dische的二苯胺（三）测定所示，这三个检测的标准曲线示于图4中 。在面板3（交流），左侧面板显示阳性/阴性对照实验中，使用适当的无功/不反应的分析物，这些可视化的结果示于原位 ，在所描述的比色皿，在协议中，1-3（以上）。右子板各自...

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

甲套件迅速判别蛋白质，RNA，DNA，并重新引入糖在潜在的异质性的生物分子样品的比色测定法进行说明。

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 ...

rapid-colorimetric-assays-to-qualitatively-distinguish-rna-dna

Research

JoVE Journal

Chemistry

41.8K 次观看.  University of Virginia. 甲套件迅速判别蛋白质，RNA，DNA，并重新引入糖在潜在的异质性的生物分子样品的比色测定法进行说明。

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

Rapid High Throughput Amylose Determination in Freeze Dried Potato Tuber Samples

This protocol describes a high through put colorimetric method that relies on the formation of a complex between iodine and chains of glucose molecules in starch. Iodine forms complexes with both amylose and long chains within amylopectin. After the addition of iodine to a starch sample, the maximum absorption of amylose and amylopectin occurs at 620 and 550 nm, respectively. The amylose/amylopectin ratio can be estimated from the ratio of the 620 and 550 nm absorbance values and comparing them to a standard curve in which specific known concentrations are plotted against absorption values. This high throughput, inexpensive method is reliable and reproducible, allowing the evaluation of large populations of potato clones.&#160;

快速高通量直链淀粉测定冻干马铃薯块茎样品

This protocol describes a high through put colorimetric method that relies on the formation of a complex between iodine and chains of glucose molecules in ...

科研

化学

Formation of Ordered Biomolecular Structures by the Self-assembly of Short Peptides

In nature, complex functional structures are formed by the self-assembly of biomolecules under mild conditions. Understanding the forces that control self-assembly and mimicking this process in vitro will bring about major advances in the areas of materials science and nanotechnology. Among the available biological building blocks, peptides have several advantages as they present substantial diversity, their synthesis in large scale is straightforward, and they can easily be modified with biological and chemical entities1,2. Several classes of designed peptides such as cyclic peptides, amphiphile peptides and peptide-conjugates self-assemble into ordered structures in solution. Homoaromatic dipeptides, are a class of short self-assembled peptides that contain all the molecular information needed to form ordered structures such as nanotubes, spheres and fibrils3-8. A large variety of these peptides is commercially available.
This paper presents a procedure that leads to the formation of ordered structures by the self-assembly of homoaromatic peptides. The protocol requires only commercial reagents and basic laboratory equipment. In addition, the paper describes some of the methods available for the characterization of peptide-based assemblies. These methods include electron and atomic force microscopy and Fourier-Transform Infrared Spectroscopy (FT-IR). Moreover, the manuscript demonstrates the blending of peptides (coassembly) and the formation of a &#34;beads on a string&#34;-like structure by this process.9 The protocols presented here can be adapted to other classes of peptides or biological building blocks and can potentially lead to the discovery of new peptide-based structures and to better control of their assembly.

This paper describes the formation of highly ordered peptide-based structures by the spontaneous process of self-assembly. The method utilizes commercially available peptides and common lab equipment. This technique can be applied to a large variety of peptides and may lead to the discovery of new peptide-based assemblies.

生物分子有序结构由短肽的自组装形成

In nature, complex functional structures are formed by the self-assembly of biomolecules under mild conditions. Understanding the forces that control ...

Confocal Imaging of Confined Quiescent and Flowing Colloid-polymer Mixtures

The behavior of confined colloidal suspensions with attractive interparticle interactions is critical to the rational design of materials for directed assembly1-3, drug delivery4, improved hydrocarbon recovery5-7, and flowable electrodes for energy storage8. Suspensions containing fluorescent colloids and non-adsorbing polymers are appealing model systems, as the ratio of the polymer radius of gyration to the particle radius&#160;and concentration of polymer control the range and strength of the interparticle attraction, respectively. By tuning the polymer properties and the volume fraction of the colloids, colloid fluids, fluids of clusters, gels, crystals, and glasses can be obtained9.&#160;Confocal microscopy, a variant of fluorescence microscopy, allows an optically transparent and fluorescent sample to be imaged with high spatial and temporal resolution in three dimensions. In this technique, a small pinhole or slit blocks the emitted fluorescent light from regions of the sample that are outside the focal volume of the microscope optical system. As a result, only a thin section of the sample in the focal plane is imaged. This technique is particularly well suited to probe the structure and dynamics in dense colloidal suspensions at the single-particle scale: the particles are large enough to be resolved using visible light and diffuse slowly enough to be captured at typical scan speeds of commercial confocal systems10. Improvements in scan speeds and analysis algorithms have also enabled quantitative confocal imaging of flowing suspensions11-16,37.&#160;In this paper, we demonstrate confocal microscopy experiments to probe the confined phase behavior and flow properties of colloid-polymer mixtures. We first prepare colloid-polymer mixtures that are density- and refractive-index matched. Next, we report a standard protocol for imaging quiescent dense colloid-polymer mixtures under varying confinement in thin wedge-shaped cells. Finally, we demonstrate a protocol for imaging colloid-polymer mixtures during microchannel flow.

Confocal microscopy is used to image quiescent and flowing colloid-polymer mixtures, which are studied as model systems for attractive suspensions. Image analysis algorithms are used to calculate structural and dynamic metrics for the colloidal particles that measure changes due to geometric confinement.

密闭静态和流动胶体聚合物混合物的共聚焦成像

The behavior of confined colloidal suspensions with attractive interparticle interactions is critical to the rational design of materials for directed ...

Preparation of Mica and Silicon Substrates for DNA Origami Analysis and Experimentation

The designed nature and controlled, one-pot synthesis of DNA origami provides exciting opportunities in many fields, particularly nanoelectronics. Many of these applications require interaction with and adhesion of DNA nanostructures to a substrate. Due to its atomically flat and easily cleaned nature, mica has been the substrate of choice for DNA origami experiments. However, the practical applications of mica are relatively limited compared to those of semiconductor substrates. For this reason, a straightforward, stable, and repeatable process for DNA origami adhesion on derivatized silicon oxide is presented here. To promote the adhesion of DNA nanostructures to silicon oxide surface, a self-assembled monolayer of 3-aminopropyltriethoxysilane (APTES) is deposited from an aqueous solution that is compatible with many photoresists. The substrate must be cleaned of all organic and metal contaminants using Radio Corporation of America (RCA) cleaning processes and the native oxide layer must be etched to ensure a flat, functionalizable surface. Cleanrooms are equipped with facilities for silicon cleaning, however many components of DNA origami buffers and solutions are often not allowed in them due to contamination concerns. This manuscript describes the set-up and protocol for in-lab, small-scale silicon cleaning for researchers who do not have access to a cleanroom or would like to incorporate processes that could cause contamination of a cleanroom CMOS clean bench. Additionally, variables for regulating coverage are discussed and how to recognize and avoid common sample preparation problems is described.

Reproducible cleaning processes for substrates used in DNA origami research are described, including bench-top RCA cleaning and derivatization of silicon oxide. Protocols for surface preparation, DNA origami deposition, drying parameters, and simple experimental set-ups are illustrated.

对于DNA折纸分析和实验准备云母和硅衬底

The designed nature and controlled, one-pot synthesis of DNA origami provides exciting opportunities in many fields, particularly nanoelectronics. Many of ...

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.

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 ...

A Method to Manipulate Surface Tension of a Liquid Metal via Surface Oxidation and Reduction

Controlling interfacial tension is an effective method for manipulating the shape, position, and flow of fluids at sub-millimeter length scales, where interfacial tension is a dominant force. A variety of methods exist for controlling the interfacial tension of aqueous and organic liquids on this scale; however, these techniques have limited utility for liquid metals due to their large interfacial tension.
Liquid metals can form soft, stretchable, and shape-reconfigurable components in electronic and electromagnetic devices. Although it is possible to manipulate these fluids via mechanical methods (e.g., pumping), electrical methods are easier to miniaturize, control, and implement. However, most electrical techniques have their own constraints: electrowetting-on-dielectric requires large (kV) potentials for modest actuation, electrocapillarity can affect relatively small changes in the interfacial tension, and continuous electrowetting is limited to plugs of the liquid metal in capillaries.
Here, we present a method for actuating gallium and gallium-based liquid metal alloys via an electrochemical surface reaction. Controlling the electrochemical potential on the surface of the liquid metal in electrolyte rapidly and reversibly changes the interfacial tension by over two orders of magnitude ( &#820;500 mN/m to near zero). Furthermore, this method requires only a very modest potential (&#60; 1 V) applied relative to a counter electrode. The resulting change in tension is due primarily to the electrochemical deposition of a surface oxide layer, which acts as a surfactant; removal of the oxide increases the interfacial tension, and vice versa. This technique can be applied in a wide variety of electrolytes and is independent of the substrate on which it rests.

We present a method to control the interfacial energy of a liquid metal in an electrolyte via electrochemical deposition (or removal) of a surface oxide layer. This simple method can control the capillary behavior of gallium-based liquid metals by tuning the interfacial energy rapidly, significantly, and reversibly using modest voltages.

方法通过表面氧化还原操纵液态金属的表面张力

Controlling interfacial tension is an effective method for manipulating the shape, position, and flow of fluids at sub-millimeter length scales, where ...

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 ...

DNA-magnetic Particle Binding Analysis by Dynamic and Electrophoretic Light Scattering

Isolation of DNA using magnetic particles is a field of high importance in biotechnology and molecular biology research. This protocol describes the evaluation of DNA-magnetic particles binding via dynamic light scattering (DLS) and electrophoretic light scattering (ELS). Analysis by DLS provides valuable information on the physicochemical properties of particles including particle size, polydispersity, and zeta potential. The latter describes the surface charge of the particle which plays major role in electrostatic binding of materials such as DNA. Here, a comparative analysis exploits three chemical modifications of nanoparticles and microparticles and their effects on DNA binding and elution. Chemical modifications by branched polyethylenimine, tetraethyl orthosilicate and (3-aminopropyl)triethoxysilane are investigated. Since DNA exhibits a negative charge, it is expected that zeta potential of particle surface will decrease upon binding of DNA. Forming of clusters should also affect particle size. In order to investigate the efficiency of these particles in isolation and elution of DNA, the particles are mixed with DNA in low pH (~6), high ionic strength and dehydration environment. Particles are washed on magnet and then DNA is eluted by Tris-HCl buffer (pH = 8). DNA copy number is estimated using quantitative polymerase chain reaction (PCR). Zeta potential, particle size, polydispersity and quantitative PCR data are evaluated and compared. DLS is an insightful and supporting method of analysis that adds a new perspective to the process of screening of particles for DNA isolation.

This protocol describes the synthesis of magnetic particles and evaluation of their DNA-binding properties via dynamic and electrophoretic light scattering. This method focuses on monitoring changes in particle size, their polydispersity and zeta potential of particle surface which play major role in binding of materials such as DNA.

动态和电泳光散射的 DNA-磁性颗粒结合分析

Isolation of DNA using magnetic particles is a field of high importance in biotechnology and molecular biology research. This protocol describes the ...

Rapid Nanoprobe Signal Enhancement by In Situ Gold Nanoparticle Synthesis

The use of nanoprobes such as gold, silver, silica or iron-oxide nanoparticles as detection reagents in bioanalytical assays can enable high sensitivity and convenient colorimetric readout. However, high densities of nanoparticles are typically needed for detection. The available synthesis-based enhancement protocols are either limited to gold and silver nanoparticles or rely on precise enzymatic control and optimization. Here, we present a protocol to enhance the colorimetric readout of gold, silver, silica, and iron oxide nanoprobes. It was observed that the colorimetric signal can be improved by up to a 10000-fold factor. The basis for such signal enhancement strategies is the chemical reduction of Au3+ to Au0. There are several chemical reactions that enable the reduction of Au3+ to Au0. In the protocol, Good&#39;s buffers and H2O2 are used and it is possible to favor the deposition of Au0 onto the surface of existing nanoprobes, in detriment of the formation of new gold nanoparticles. The protocol consists of the incubation of the microarray with a solution consisting of chloroauric acid and H2O2 in 2-(N-morpholino)ethanesulfonic acid pH 6 buffer following the nanoprobe-based detection assay. The enhancement solution can be applied to paper and glass-based sensors. Moreover, it can be used in commercially available immunoassays as demonstrated by the application of the method to a commercial allergen microarray. The signal development requires less than 5 min of incubation with the enhancement solution and the readout can be assessed by naked eye or low-end image acquisition devices such as a table-top scanner or a digital camera.

Rapid Nanoprobe Signal Enhancement by In Situ Gold Nanoparticle Synthesis

In this work, a protocol for signal enhancement of nanoprobe-based biosensing is presented. The protocol is based on the reduction of chloroauric acid onto the surface of existing nanoprobes that consist of gold, silver, silica or iron-oxide nanoparticles.

原位金纳米粒子合成快速 Nanoprobe 信号增强

The use of nanoprobes such as gold, silver, silica or iron-oxide nanoparticles as detection reagents in bioanalytical assays can enable high sensitivity ...

Solvothermal Synthesis of MIL-96 and UiO-66-NH2 on Atomic Layer Deposited Metal Oxide Coatings on Fiber Mats

Metal-organic frameworks (MOFs), which contain reactive metal clusters and organic ligands allowing for large porosities and surface areas, have proven effective in gas adsorption, separations, and catalysis. MOFs are most commonly synthesized as bulk powder, requiring additional processes to adhere them to functional devices and fabrics that risk decreasing the powder porosity and adsorption capacity. Here, we demonstrate a method of first coating fabrics with metal oxide films using atomic layer deposition (ALD). This process creates conformal films of controllable thickness on each fiber, while providing a more reactive surface for MOF nucleation. By submerging the ALD coated fabric in solution during solvothermal MOF synthesis, the MOFs create a conformal, well-adhered coating on the fibers, resulting in a MOF-functionalized fabric, without additional adhesion materials that may block MOF pores and functional sites. Here we demonstrate two solvothermal synthesis methods. First, we form a MIL-96(Al) layer on polypropylene fibers using synthetic conditions that convert the metal oxide to MOF. Using initial inorganic films of varying thicknesses, diffusion of the organic linker into the inorganic allows us to control the extent of MOF loading on the fabric. Second, we perform a solvothermal synthesis of UiO-66-NH2 in which the MOF nucleates on the conformal metal oxide coating on polyamide-6 (PA-6) fibers, thereby producing a uniform and conformal thin film of MOF on the fabric. The resulting materials can be directly incorporated into filter devices or protective clothing and eliminate the maladroit qualities of loose powder.

Solvothermal Synthesis of MIL-96 and UiO-66-NH2 on Atomic Layer Deposited Metal Oxide Coatings on Fiber Mats

Metal-organic frameworks are effective in gas storage and heterogeneous catalysis, but typical synthesis methods result in loose powders that are difficult to incorporate into smart materials. We demonstrate a method of first coating fabrics with ALD metal oxides, resulting in conformal films of MOF on the fabrics during solvothermal synthesis.

溶剂热合成 MIL-96 和 UiO-66-NH2在纤维垫上原子层沉积金属氧化物涂层上的合成

Metal-organic frameworks (MOFs), which contain reactive metal clusters and organic ligands allowing for large porosities and surface areas, have proven ...