这项工作是由国家科学基金会奖＃0920274和南卡罗来纳州试验站项目（SC - 1700340）幼稚园资助计划的支持。本文是技术贡献的克莱姆森大学实验站5929号。

此协议的总体方案概述图1。 1。标准曲线和检测解决方案的准备工作<ol><li>准备100毫升2 mol / L的羟胺盐酸溶液。称取13.9克盐酸羟胺（兆瓦69.49克/摩尔），溶于约50毫升蒸馏水，去离子水（DDH 2 O）的。使用氢氧化钾颗粒或浓缩液，调整pH至7.0。带来的最终体积至100 mL。该解决方案可在常温下保存长达30天，或在4 ° C下90天。 </li><li>准备一个三氯化铁/盐酸溶液100毫升。称取13.5克氯化铁（兆瓦270.32克/摩尔），溶于约50毫升DDH 2 O添加41.3毫升的浓盐酸（12.1 mol / L的），使终体积为100 mL。氯化铁的终浓度0.5 mol / L的终浓度的盐酸，将5 MOL/ L。解决的办法是在常温下保存。 </li><li>准备100毫升trichoroacetic酸了1.84 mol / L的解决方案。称取30.0克的三氯乙酸（兆瓦163.39克/摩尔），溶于DDH 2 O，使最终体积至100 mL。解决的办法是在常温下保存。 </li><li>准备0.1 mol / L的乙酰，磷酸盐溶液5毫升。称取0.092克乙酰磷酸（兆瓦184.06克/摩尔），溶于DDH 2 O，使最终体积至5 mL。分装的解决方案，离心管（0.25毫升每管），并在-20℃冻结，直到使用。解冻的解决方案应放在冰，可用于约1小时。乙酰磷酸解决方案已经解冻的，不应该再冷冻和再利用。 </li><li>准备5毫升0.1 mol / L的ADP的解决方案。称取0.24克（兆瓦472.5克/摩尔）的ADP，溶于DDH 2 O，并把最终体积至5 mL。分装在离心管（每管0.5毫升）的解决方案，并冻结在-20 ° C直到使用。保持解冻冰上直至使用的解决方案。 </li><li>准备50毫升的1 mol / L的三解决方案。称取6.06克三（兆瓦121.14克/摩尔），溶于40毫升DDH 2 O溶液的pH值调整到7.0用盐酸和带来最终容至50 ml。在室温下的存储解决方案。 </li><li>准备1 mol / L的氯化镁溶液10毫升。称取2.03克氯化镁（兆瓦203.31克/摩尔），溶于DDH 2 O，并把最后容至10 mL。储存在室温下的解决方案。 </li><li>准备好三氯化铁/盐酸溶液和三氯乙酸溶液等体积混合25毫升的开发解决方案。开发解决方案，应准备新鲜的每一天，在室温下储存。 </li></ol> 2。乙酰磷酸标准曲线的制备<ol><li>乙酰磷酸标准曲线生成乙酰磷酸已知金额。标准曲线的6至eighT分是合适的。该标准的范围应该每300μL，这与0.1〜3 mmol / L的终浓度从0.03μmoles到0.9μmoles稀乙酰磷酸原液100毫米三在终体积为300μL的解决方案，在适当的浓度。还应该准备一个没有控制乙酰磷酸样本。 </li><li>每个标准控制在37 ° C的热块1分钟。 </li><li>添加50μL盐酸羟胺溶液到每一个标准和晃动三次，控制和混合。放入60 ° C的热块的标准，并孵育5分钟。 </li><li>加入100μL的开发解决方案的标准和控制，通过摇动三次拌匀，并让颜色在室温下至少一分钟的发展。 </li><li>离心机中离心1分钟18000 XG的所有标准和控制。 </li><li>分光光度计一个测量样品吨540 nm处使用1毫升塑料试管，用分光空白的控制。 </li><li>使用适当的数据分析和绘图软件（如Kaleidagraph，协同软件）生成一个标准的曲线。 R 2的值大于或等于0.99是可取的。数据绘制与乙酰磷酸目前μmoles，在540 nm处的吸光度。 </li></ol> 3。醋酸激酶的活性含量为<ol><li>使用所述的解决方案，准备一个反应混合物中包含终浓度为0.1 mol / L的Tris缓冲液，10 mmol / L的氯化镁，5 mmol / L的ADP，和2 mmol / L的乙酰磷酸。在10 mL反应混合物将需要8.2毫升DDH 2 O，1.0毫升三的解决方案，为0.1 mL氯化镁溶液，0.5毫升ADP的解决方案，0.2毫升乙酰磷酸盐溶液。分布在300μL分装离心管的反应混合。动力学参数和底物浓度的优化的测定，一个基板可以多样和其他在恒定浓度的反应举行。 </li><li>预1分钟的反应在37 ° C的热块。 </li><li>添加酶反应所需的量，并立即返回到37℃的热块管。还包括一个控制反应没有酶添加。如果进行几个反应管，在特定的时间间隔，添加酶。一定要跟踪时间。 （注：酶的使用量将需要优化，以确保乙酰磷酸或ADP基板都不会枯竭 - 见讨论部分） </li><li> 5分钟后，加入盐酸羟胺溶液50μL，每个反应和控制，混合，放置在60 ° C的热块管。如果正在执行的一些反应，这应该在相同的时间间隔添加酶。 </li><li> 5分钟的反应和控制孵育60 ° C。 </li><li>新增100＆万亩; L的开发解决方案，每个反应和控制，混合，在实验室工作台上的试管架。允许发展至少1分钟的颜色。 </li><li>离心机中离心1分钟18000 XG管。 </li><li>分光光度计在540 nm，使用1毫升的塑料试管和乙酰磷酸标准曲线进行比较，以确定乙酰磷酸目前量的测量样品。 </li><li>乙酰磷酸耗尽基板量可以由乙酰磷酸减去从量控制缺乏酶的乙酰磷酸酶反应后的剩余金额。使用适当的数据和图形软件（例如Kaleidagraph，协同软件）进行数据分析。 R 2的值比0.97更大的是可取的。 </li></ol> 4。代表性的成果： 这个实验的目的是直接测量醋酸激酶活性离子的醋酸形成。这是通过测量乙酰磷酸基板的消费，因为是不容易直接测量，醋酸生产的。乙酰磷酸酶反应过程中给定的时间后剩余是转化为乙酰氧肟，并在其后铁氧肟复杂，其中有一个颜色偏红（图2），可在540 nm处测定。标准曲线的绘制吸光度与μmoles标准的乙酰磷酸目前用于确定每个反应中剩余的乙酰磷酸量。标准曲线应通过零点的线性。一个有代表性的标准曲线如图3所示的1.2332％μmole乙酰磷酸目前和 R 2值0.99吸光度单位，表明该数据符合线性方程的斜率。标准曲线数据的线性拟合方程用于计算乙酰磷酸反应中剩余的数量。该μmoles乙酰消耗磷酸是在目前缺乏酶和乙酰磷酸酶反应后剩余的μmoles控制反应的乙酰磷酸μmoles之间的差异决定的。 纯化，重组M。嗜热醋酸激酶检测使用所描述的协议。如图4所示的实验，ADP浓度保持不变在5 mmol / L的浓度变化的反应乙酰磷酸乙酰磷酸，以确定的 K m。正如所料，这个实验表明，这种酶如下每个基板标准米氏般的动力学，并产生了双曲饱和曲线在策划活动与乙酰磷酸盐浓度。明显的 K m值乙酰磷酸为0.27 ± 0.01 mmol / L时相比，毫不逊色出版的 K m值0.47 ± 0.01 mmol / L的使用加上作为说13。 直接分析，也可以用来衡量利用衬底比ATP，这是不可能使用标准的耦合检测的醋酸激酶的活动。纯化的重组大肠杆菌阿米巴醋酸激酶16，PP 我 ，而不是ATP的产生在醋酸形成的方向，受到这个实验中，使用ADP的地方磷酸钠。反应磷酸钠的浓度保持不变，在0.2 mol / L的乙酰磷酸盐浓度是不同的。至于的M.嗜热醋酸激酶， 大肠杆菌阿米巴醋酸激酶遵循米氏般的动力学和乙酰磷酸双曲饱和曲线（图5）。明显的 K m值乙酰磷酸为0.50 ± 0.006毫摩尔/升。 Reeves和格思里15如何确定的 K m值的0.06毫摩尔/大号原酶的乙酰磷酸;<html>以往，他们的酶只是部分纯化和检测涉及四个耦合的酶，也只有部分纯化。因此，它是很难直接比较这些值。 协议计划<ol><li>生成乙酰磷酸标准曲线</li><li>稀释到所需浓度的酶</li><li>准备反应混合物和分装300μL离心管</li><li> Preinc​​ubate反应在37 ° C为1分钟</li><li>启动通过添加酶在特定的时间间隔反应</li><li>添加羟胺在相同的时间间隔，停止反应，并培育在60℃，5分钟</li><li>添加氯化铁/三氯乙酸溶液显色</li><li>离心管18,000 1分钟XG </li><li>在540 nm处测量吸光度</li><li>乙酰消耗磷矿量确定标准曲线比较</li></ol>tp_upload/3474/3474fig1.jpg“/&gt; 图1。议定书“的计划 <img alt="图2" src="/files/ftp_upload/3474/3474fig2.jpg" /> 图2。乙酰磷酸标准 。乙酰磷酸浓度增加的反应，羟胺和颜色所述。缺乏乙酰磷酸的控制是亮黄色，并含有对乙酰磷酸越来越多的反应都先后在颜色较深。这种颜色的变化是在540 nm处测量。 <img alt="图3" src="/files/ftp_upload/3474/3474fig3.jpg" /> 图3。乙酰磷酸样品标准曲线。与羟胺反应的乙酰磷酸数额不等，颜色开发解决方案和吸光度在540 nm处测定。与μmoles乙酰磷酸在540 nm的吸光度，绘制线性拟合的数据应用。/ P&gt; <img alt="图4" src="/files/ftp_upload/3474/3474fig4.jpg" /> 图4 ATP生产M.乙酰磷酸的利用。 嗜热醋酸激酶 。酶活性测定醋酸形成中存在的5 mmol / L的ADP与不同浓度的乙酰磷酸的方向。乙酰磷酸消耗金额被认定为起点金额乙酰磷酸反应和乙酰磷酸酶反应后剩余的金额之间的差额。检测显示，错误的酒吧一式三份。 <img alt="图5" src="/files/ftp_upload/3474/3474fig5.jpg" /> 图5。E.利用由PP 我生产的乙酰磷酸阿米巴醋酸激酶 。在醋酸形成的方向酶活性测定在0的存在。2 mol / L的不同浓度的乙酰磷酸磷酸钠。乙酰磷酸消耗金额被认定为起点金额乙酰磷酸反应和乙酰磷酸酶反应后剩余的金额之间的差额。检测显示，错误的酒吧一式三份。

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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+continuous+assay+of+acetate+kinase+activity:+measurement+of+inorganic+phosphate+release+generated+by+hydroxylaminolysis+of+acetyl+phosphate.">A continuous assay of acetate kinase activity: measurement of inorganic phosphate release generated by hydroxylaminolysis of acetyl phosphate.</a> Bioorg. Chem. 36, 65-69 (2008).</li><li>Nakajima, H., Suzuki, K., Imahori, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purification+and+properties+of+acetate+kinase+from+Bacillus+stearothermophilus.">Purification and properties of acetate kinase from Bacillus stearothermophilus.</a> J. Biochem. 84, 193-203 (1978).</li></ol>

没有利益冲突的声明。

在这个实验中的乙酰磷酸的检测是根据足够浓度的盐酸羟胺和氯化铁溶液的浓度和酸度依赖。更改的检测量将需要重新考虑这两个组件。酶促反应这里描述的是37 °与5分钟，在60 ° C时，羟胺终止。这较高的温度是至关重要的，以便其余乙酰磷酸迅速转化为乙酰氧肟。颜色一步发展和吸光度的测量时间是不太重要的，羟胺溶液此外，可以在短短的5分钟至15分钟进行。样品应该离心读取吸光度之前，...

<table border="1"><tr><td width="160"> 试剂名称 </td><td width="108"> 公司 </td><td width="134"> 目录编号 </td><td width="103"> 评论 </td></tr><tr><td>乙酰磷酸</td><td>西格玛爱秩序</td><td> 01409 </td><td>锂盐（97％） </td></tr><tr><td>钠磷酸二氢（脱水） </td><td> ThermoFisher </td><td> S381 </td><td></td></tr><tr><td>钠磷酸氢二钠（无水） </td><td> ThermoFisher </td><td> S374 </td><td></td></tr><tr><td>氯化镁（六水） </td><td> ThermoFisher </td><td> M33 </td><td></td></tr><tr><td> Tris碱</td><td> ThermoFisher </td><td> B152 </td><td></td></tr><tr><td>三氯化铁（六水） </td><td> ThermoFisher </td><td> I88 </td><td></td></tr><tr><td>三氯乙酸</td><td> ThermoFisher </td><td> A324 </td><td></td></tr><tr><td>盐酸羟胺</td><td> ThermoFisher </td><td> H330 </td><td></td></tr><tr><td>腺苷5&#39; -二磷酸钠盐</td><td>西格玛爱秩序</td><td> A2754 </td><td></td></tr><tr><td> Biomate三分光光度计</td><td> ThermoFisher </td><td> 142982082 </td><td>标准紫外/可见分光光度计</td></tr></table>

direct detection of the acetate-forming activity  of the enzyme acetate kinase

醋酸激酶，醋酸盐和糖激酶- HSP70 -肌动蛋白（ASKHA）1-5酶超家族成员，负责对醋酸的可逆磷酸化乙酰磷酸盐作为基质利用ATP 。醋酸激酶是无处不在的细菌 ，发现一个古属的，而且也存在于 真核生物 6微生物。最特征的醋酸激酶是由产甲烷古菌甲烷嗜 热 7-14 。 阿米巴 ，阿米巴痢疾的病原体已被隔离醋酸激酶只能利用聚丙烯 ，但不乙酰磷酸形成的方向ATP，并迄今只发现该属15,16。 在乙酰磷酸形成的方向，醋酸激酶活性通常是测量使用的氧肟检测，首先由李普曼描述17-20日 ，耦合检测的ATP转化为ADP耦合NADH氧化为NAD +的酶丙酮酸激酶和乳酸脱氢酶21,22，或乙酰磷酸盐产品的反应后释放的无机磷的实验测量羟胺23。醋酸形成方向相反的活动，是衡量耦合ATP的形成，从ADP 减少NADP +，NADPH酶己糖激酶和葡萄糖-6 - 磷酸脱氢酶24。 在这里，我们描述了醋酸醋酸形成，不需要耦合酶的方向激酶活性检测的方法，而是被直接测定乙酰磷酸消费。酶反应后，剩余的乙酰磷酸转化为羟肟检测，可以测量分光光度计的一个铁氧肟复杂。因此，不像STandard加上这个方向是依赖于从ADP ATP的生产检测，这种直接的检测可用于醋酸激酶产生ATP或PP 我 。

Watch this Scientific Journal Video about Direct Detection of the Acetate-forming Activity  of the Enzyme Acetate Kinase at JoVE.com

Direct Detection of the Acetate-forming Activity  of the Enzyme Acetate Kinase

醋酸激酶活性测定方法的描述。本实验利用一个直接反应在醋酸形成的方向确定不同磷受体酶的活性和乙酸激酶动力学。此外，这种方法可以用于化验等乙酰磷酸盐或乙酰CoA利用酶。

醋酸形成醋酸酶激酶活性的直接检测

Acetate kinase, a member of the acetate and sugar kinase-Hsp70-actin (ASKHA) enzyme superfamily1-5, is responsible for the reversible phosphorylation of ...

direct-detection-acetate-forming-activity-enzyme-acetate

Research

JoVE Journal

Biology

23.0K 次观看.  Clemson University. 醋酸激酶活性测定方法的描述。本实验利用一个直接反应在醋酸形成的方向确定不同磷受体酶的活性和乙酸激酶动力学。此外，这种方法可以用于化验等乙酰磷酸盐或乙酰CoA利用酶。

视频: Direct Detection of the Acetate-forming Activity  of the Enzyme Acetate Kinase

Detection of Protein Ubiquitination

Ubiquitination, the covalent attachment of the polypeptide ubiquitin to target proteins, is a key posttranslational modification carried out by a set of three enzymes. They include ubiquitin-activating enzyme E1, ubiquitin-conjugating enzyme E2, and ubiquitin ligase E3. Unlike to E1 and E2, E3 ubiquitin ligases display substrate specificity. On the other hand, numerous deubiquitylating enzymes have roles in processing polyubiquitinated proteins. Ubiquitination can result in change of protein stability, cellular localization, and biological activity. Mutations of genes involved in the ubiquitination/deubiquitination pathway or altered ubiquitin system function are associated with many different human diseases such as various types of cancer, neurodegeneration, and metabolic disorders. The detection of altered or normal ubiquitination of target proteins may provide a better understanding on the pathogenesis of these diseases. &nbsp;Here, we describe protocols to detect protein ubiquitination in cultured cells in vivo and test tubes in vitro. These protocols are also useful to detect other ubiquitin-like small molecule modification such as sumolyation and neddylation.

Ubiquitination is a key posttranslational modification carried out by a set of three enzymes. Mutations of genes involved in this modification are associated with many different human diseases. Here, we describe protocols to detect protein ubiquitination in cultured cells in vivo and test tubes in vitro.

蛋白泛素化的检测

Ubiquitination, the covalent attachment of the polypeptide ubiquitin to target proteins, is a key posttranslational modification carried out by a set of ...

科研

生物学

Use of the Protease Fluorescent Detection Kit to Determine Protease Activity

The Protease Fluorescent Detection Kit provides ready-to-use reagents for detecting the presence of protease activity. This simple assay to detect protease activity uses casein labeled with fluorescein isothiocyanate (FITC) as the substrate.
Protease activity results in the cleavage of the FITC-labeled casein substrate into smaller fragments, which do not precipitate under acidic conditions. After incubation of the protease sample and substrate, the reaction is acidified with the addition of trichloroacetic acid (TCA). The mixture is then centrifuged with the undigested substrate forming a pellet and the smaller, acid soluble fragments remaining in solution. The supernatant is neutralized and the fluorescence of the FITC-labeled fragments is measured.
The described kit procedure detects the trypsin protease control at a concentration of approximately 0.5 &mu;g/ml (5 ng of trypsin added to the assay). This sensitivity can be increased with a longer incubation time, up to 24 hours. The assay is performed in microcentrifuge tubes and procedures are provided for fluorescence detection using either cuvettes or multiwell plates.

The Protease Fluorescent Detection Kit is designed for the measurement of protease activity using fluorometry. It is also suitable for detection of trace amounts of protease contamination. The method is based on the proteolytic hydroysis of a proprietary formulation of a FITC-labeled casein substrate.

蛋白酶荧光检测试剂盒，用它来确定蛋白酶活性

The Protease Fluorescent Detection Kit provides ready-to-use reagents for detecting the presence of protease activity. This simple assay to detect ...

ELIME (Enzyme Linked Immuno Magnetic Electrochemical) Method for Mycotoxin Detection

Immunoassays are a valid alternative to the more expensive and time consuming quantitative HPLC or GC1, 2 methods for the screening detection of hazardous mycotoxins in food commodities. In this protocol we show how to fabricate and interrogate an electrochemical competitive Enzyme linked immunomagnetic assay based on the use of magnetic beads as solid support for the immunochemical chain3 and screen printed electrodes as sensing platform.
Our method aims to determine the total amount of HT-2 and T-2 toxins, mycotoxins belonging to the trichothecenes family and of great concern for human health4. The use of an antibody clone with a cross reactivity of 100% towards HT-2 and T-2 allows to simultaneously detect both toxins with similar sensitivity5.
The first step of our assay is the coating step where we immobilize HT2-KLH conjugate toxin on the surface of magnetic beads. After a blocking step, necessary to avoid non-specific absorptions, the addition of a monoclonal antibody allows the competition between immobilized HT-2 and free HT-2 or T-2 present in the sample or dissolved in a standard solution.
At the end of the competition step, the amount of monoclonal antibody linked to the immobilized HT-2 will be inversely proportional to the amount of toxin in the sample solution.
A secondary antibody labeled with alkaline phosphatase (AP) is used to reveal the binding between the specific antibody and the immobilized HT-2. The final measurement step is performed by dropping an aliquot of magnetic bead suspension, corresponding to a specific sample/standard solution, on the surface of a screen-printed working electrode; magnetic beads are immobilized and concentrated by means of a magnet placed precisely under the screen-printed electrode. After two minutes of incubation between magnetic beads and a substrate for AP, the enzymatic product is detected by Differential Pulse Voltammetry (DPV) using a portable instrument (PalmSens) also able to initiate automatically eight measurements within an interval of few seconds.

ELIME Enzyme Linked Immuno Magnetic Electrochemical Method for Mycotoxin Detection

A protocol to detect trichothecenes (mycotoxins of concern for human health) using a newly developed screening method based on a competitive immunochemical method and a final electrochemical detection is demonstrated.

ELIME霉菌毒素的检测方法（酶联免疫磁电化学）

Immunoassays are a valid alternative to the more expensive and time consuming quantitative HPLC or GC1, 2 methods for the screening detection of hazardous ...

Production of Transgenic Xenopus laevis by Restriction Enzyme Mediated Integration and Nuclear Transplantation

Stable integration of cloned gene products into the Xenopus genome is necessary to control the time and place of expression, to express genes at later stages of embryonic development, and to define how enhancers and promoters regulate gene expression within the embryo. The protocol demonstrated here can be used to efficiently produce transgenic Xenopus laevis embryos. This transgenesis approach involves three parts: 1. Sperm nuclei are isolated from adult X. laevis testis by treatment with lysolecithin, which permeabilizes the sperm plasma membrane. 2. Egg extract is prepared by low speed centrifugation, addition of calcium to cause the extract to progress to interphase of the cell cycle, and a high-speed centrifugation to isolate interphase cytosol. 3. Nuclear transplantation: the nuclei and extract are combined with the linearized plasmid DNA to be introduced as the transgene and a small amount of restriction enzyme. During a short reaction, egg extract partially decondenses the sperm chromatin and the restriction enzyme generates chromosomal breaks that promote recombination of the transgene into the genome. The treated sperm nuclei are then transplanted into unfertilized eggs. Integration of the transgene usually occurs prior to the first embryonic cleavage such that the resulting embryos are not chimeric. These embryos can be analyzed without any need to breed to the next generation, allowing for efficient and rapid generation of transgenic embryos for analyses of promoter and gene function. Adult X. laevis resulting from this procedure also propagate the transgene through the germline and can be used to generate lines of transgenic animals for multiple purposes.

Production of Transgenic Xenopus laevis by Restriction Enzyme Mediated Integration and Nuclear Transplantation

This video protocol demonstrates a method for generating transgenic Xenopus laevis by introduction of transgenes into sperm nuclei followed by nuclear transplantation into unfertilized eggs.

生产转基因非洲爪蟾限制性内切酶介导的整合和核移植

Stable integration of cloned gene products into the Xenopus genome is necessary to control the time and place of expression, to express genes at later ...

Detection of Nitric Oxide and Superoxide Radical Anion by Electron Paramagnetic Resonance Spectroscopy from Cells using Spin Traps

Reactive nitrogen/oxygen species (ROS/RNS) at low concentrations play an important role in regulating cell function, signaling, and immune response but in unregulated concentrations are detrimental to cell viability1, 2. While living systems have evolved with endogenous and dietary antioxidant defense mechanisms to regulate ROS generation, ROS are produced continuously as natural by-products of normal metabolism of oxygen and can cause oxidative damage to biomolecules resulting in loss of protein function, DNA cleavage, or lipid peroxidation3, and ultimately to oxidative stress leading to cell injury or death4. 
Superoxide radical anion (O2&bull;-) is the major precursor of some of the most highly oxidizing species known to exist in biological systems such as peroxynitrite and hydroxyl radical. The generation of O2&bull;- signals the first sign of oxidative burst, and therefore, its detection and/or sequestration in biological systems is important. In this demonstration, O2&bull;- was generated from polymorphonuclear neutrophils (PMNs). Through chemotactic stimulation with phorbol-12-myristate-13-acetate (PMA), PMN generates O2&bull;- via activation of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase5. 
Nitric oxide (NO) synthase which comes in three isoforms, as inducible-, neuronal- and endothelial-NOS, or iNOS, nNOS or eNOS, respectively, catalyzes the conversion of L- arginine to L-citrulline, using NADPH to produce NO6. Here, we generated NO from endothelial cells. Under oxidative stress conditions, eNOS for example can switch from producing NO to O2&bull;- in a process called uncoupling, which is believed to be caused by oxidation of heme7 or the co-factor, tetrahydrobiopterin (BH4)8.
There are only few reliable methods for the detection of free radicals in biological systems but are limited by specificity and sensitivity. Spin trapping is commonly used for the identification of free radicals and involves the addition reaction of a radical to a spin trap forming a persistent spin adduct which can be detected by electron paramagnetic resonance (EPR) spectroscopy. The various radical adducts exhibit distinctive spectrum which can be used to identify the radicals being generated and can provide a wealth of information about the nature and kinetics of radical production9.
The cyclic nitrones, 5,5-dimethyl-pyrroline-N-oxide, DMPO10, the phosphoryl-substituted DEPMPO11, and the ester-substituted, EMPO12 and BMPO13, have been widely employed as spin traps--the latter spin traps exhibiting longer half-lives for O2&bull;- adduct. Iron (II)-N-methyl-D-glucamine dithiocarbamate, Fe(MGD)2 is commonly used to trap NO due to high rate of adduct formation and the high stability of the spin adduct14.

Electron paramagnetic resonance (EPR) spectroscopy was employed to detect nitric oxide from bovine aortic endothelial cells and superoxide radical anion from human neutrophils using iron (II)-N-methyl-D-glucamine dithiocarbamate, Fe(MGD)2 and 5,5-dimethyl-1-pyroroline-N-oxide, DMPO, respectively.

从细胞中一氧化氮和超氧负离子自由基的电子顺磁共振光谱的检测，使用自旋陷阱

Reactive nitrogen/oxygen species (ROS/RNS) at low concentrations play an important role in regulating cell function, signaling, and immune response but in ...

Monitoring Kinase and Phosphatase Activities Through the Cell Cycle by Ratiometric FRET

F&ouml;rster resonance energy transfer (FRET)-based reporters1 allow the assessment of endogenous kinase and phosphatase activities in living cells. Such probes typically consist of variants of CFP and YFP, intervened by a phosphorylatable sequence and a phospho-binding domain. Upon phosphorylation, the probe changes conformation, which results in a change of the distance or orientation between CFP and YFP, leading to a change in FRET efficiency (Fig 1). Several probes have been published during the last decade, monitoring the activity balance of multiple kinases and phosphatases, including reporters of PKA2, PKB3, PKC4, PKD5, ERK6, JNK7, Cdk18, Aurora B9 and Plk19. Given the modular design, additional probes are likely to emerge in the near future10. 
Progression through the cell cycle is affected by stress signaling pathways 11. Notably, the cell cycle is regulated differently during unperturbed growth compared to when cells are recovering from stress12.Time-lapse imaging of cells through the cell cycle therefore requires particular caution. This becomes a problem particularly when employing ratiometric imaging, since two images with a high signal to noise ratio are required to correctly interpret the results. Ratiometric FRET imaging of cell cycle dependent changes in kinase and phosphatase activities has predominately been restricted to sub-sections of the cell cycle8,9,13,14.
Here, we discuss a method to monitor FRET-based probes using ratiometric imaging throughout the human cell cycle. The method relies on equipment that is available to many researchers in life sciences and does not require expert knowledge of microscopy or image processing.

FRET-based reporters are increasingly used to monitor kinase and phosphatase activities in live cells. Here we describe a method on how to use FRET-based reporters to assess cell cycle-dependent changes in target phosphorylation.

通过细胞周期激酶和磷酸酶活性成比例的FRET监测

F&ouml;rster resonance energy transfer (FRET)-based reporters1 allow the assessment of endogenous kinase and phosphatase activities in living cells. Such ...

Assaying the Kinase Activity of LRRK2 in vitro

Leucine Rich Repeat Kinase 2 (LRRK2) is a 2527 amino acid member of the ROCO family of proteins, possessing a complex, multidomain structure including a GTPase domain (termed ROC, for Ras of Complex proteins) and a kinase domain1. The discovery in 2004 of mutations in LRRK2 that cause Parkinson's disease (PD) resulted in LRRK2 being the focus of a huge volume of research into its normal function and how the protein goes awry in the disease state2,3. Initial investigations into the function of LRRK2 focused on its enzymatic activities4-6. Although a clear picture has yet to emerge of a consistent alteration in these due to mutations, data from a number of groups has highlighted the importance of the kinase activity of LRRK2 in cell death linked to mutations7,8. Recent publications have reported inhibitors targeting the kinase activity of LRRK2, providing a key experimental tool9-11. In light of these data, it is likely that the enzymatic properties of LRRK2 afford us an important window into the biology of this protein, although whether they are potential drug targets for Parkinson's is open to debate.
A number of different approaches have been used to assay the kinase activity of LRRK2. Initially, assays were carried out using epitope tagged protein overexpressed in mammalian cell lines and immunoprecipitated, with the assays carried out using this protein immobilised on agarose beads4,5,7. Subsequently, purified recombinant fragments of LRRK2 in solution have also been used, for example a GST tagged fragment purified from insect cells containing residues 970 to 2527 of LRRK212. Recently, Dani&euml;ls et al. reported the isolation of full length LRRK2 in solution from human embryonic kidney cells, however this protein is not widely available13. In contrast, the GST fusion truncated form of LRRK2 is commercially available (from Invitrogen, see table 1 for details), and provides a convenient tool for demonstrating an assay for LRRK2 kinase activity. Several different outputs for LRRK2 kinase activity have been reported. Autophosphorylation of LRRK2 itself, phosphorylation of Myelin Basic Protein (MBP) as a generic kinase substrate and phosphorylation of an artificial substrate - dubbed LRRKtide, based upon phosphorylation of threonine 558 in Moesin - have all been used, as have a series of putative physiological substrates including &alpha;-synuclein, Moesin and 4-EBP14-17. The status of these proteins as substrates for LRRK2 remains unclear, and as such the protocol described below will focus on using MBP as a generic substrate, noting the utility of this system to assay LRRK2 kinase activity directed against a range of potential substrates.

Assaying the Kinase Activity of LRRK2 in vitro

Leucine Rich Repeat Kinase 2 is a large multidomain kinase, mutations in which are the most common genetic cause of Parkinson's disease. Analysis of the kinase activity of this protein has proven to be a crucial tool in understanding the biology and dysfunction of this protein. In this paper, in vitro assaying of the kinase activity of LRRK2 and a selection of its mutants is described, providing an experimental system to examine phosphorylation of putative substrates and potential dysfunction of LRRK2 in disease.

含量测定的LRRK2的激酶活性在体外</em

Leucine Rich Repeat Kinase 2 (LRRK2) is a 2527 amino acid member of the ROCO family of proteins, possessing a complex, multidomain structure including a ...

GC-based Detection of Aldononitrile Acetate Derivatized Glucosamine and Muramic Acid for Microbial Residue Determination in Soil

Quantitative approaches to characterizing microorganisms are crucial for a broader understanding of the microbial status and function within ecosystems. Current strategies for microbial analysis include both traditional laboratory culture-dependent techniques and those based on direct extraction and determination of certain biomarkers1, 2. Few among the diversity of microbial species inhabiting soil can be cultured, so culture-dependent methods introduce significant biases, a limitation absent in biomarker analysis.
The glucosamine, mannosamine, galactosamine and muramic acid have been well served as measures of both the living and dead microbial mass, of these the glucosamine (most abundant) and muramic acid (uniquely from bacterial cell) are most important constituents in the soil systems3, 4. However, the lack of knowledge on the analysis restricts the wide popularization among scientific peers. Among all existing analytical methods, derivatization to aldononitrile acetates followed by GC-based analysis has emerged as a good option with respect to optimally balancing precision, sensitivity, simplicity, good chromatographic separation, and stability upon sample storage5.
Here, we present a detailed protocol for a reliable and relatively simple analysis of glucosamine and muramic acid from soil after their conversion to aldononitrile acetates. The protocol mainly comprises four steps: acid digestion, sample purification, derivatization and GC determination. The step-by-step procedure is modified according to former publications6, 7. In addition, we present a strategy to structurally validate the molecular ion of the derivative and its ion fragments formed upon electron ionization. We applied GC-EI-MS-SIM, LC-ESI-TOF-MS and isotopically labeled reagents to determine the molecular weight of aldononitrile acetate derivatized glucosamine and muramic acid; we used the mass shift of isotope-labeled derivatives in the ion spectrum to investigate ion fragments of each derivatives8. In addition to the theoretical elucidation, the validation of molecular ion of the derivative and its ion fragments will be useful to researchers using &delta;13C or ion fragments of these biomarkers in biogeochemical studies9, 10.

We describe a method protocol for the GC-based analysis of the aldonitrile acetate derivatives of glucosamine and muramic acid extracted from soil. For elucidation of the chemical mechanism, we also present a strategy to confirm the structure of the derivative and the ion fragments formed upon electron ionization.

衍生Aldononitrile乙酸氨基葡萄糖和胞壁酸的微生物在土壤中残留量的测定气相色谱的检测

Quantitative approaches to characterizing microorganisms are crucial for a broader understanding of the microbial status and function within ecosystems. ...

Measurement of Larval Activity in the Drosophila Activity Monitor

Drosophila larvae are used in many behavioral studies, yet a simple device for measuring basic parameters of larval activity has not been available. This protocol repurposes an instrument often used to measure adult activity, the TriKinetics Drosophila activity monitor (MB5 Multi-Beam Activity Monitor) to study larval activity. The instrument can monitor the movements of animals in 16 individual 8 cm glass assay tubes, using 17 infrared detection beams per tube. Logging software automatically saves data to a computer, recording parameters such as number of moves, times sensors were triggered, and animals&#8217; positions within the tubes. The data can then be analyzed to represent overall locomotion and/or position preference as well as other measurements. All data are easily accessible and compatible with basic graphing and data manipulation software. This protocol will discuss how to use the apparatus, how to operate the software and how to run a larval activity assay from start to finish.

Measurement of Larval Activity in the Drosophila Activity Monitor

This report describes a method for measuring Drosophila larval activity using the TriKinetics Drosophila Activity Monitor. The device employs infrared beams to detect movements of up to 16 individual animals. Data can be analyzed to represent motion parameters including rates and the positions of the animals within the assay chambers.

幼虫活动的测量<em&gt;果蝇</em&gt;活动监视器

Drosophila larvae are used in many behavioral studies, yet a simple device for measuring basic parameters of larval activity has not been available. This ...

Studying the Activity of Neuropeptides and Other Regulators of the Excretory System in the Adult Mosquito

Studies of insect physiology, particularly in those species that are vectors of pathogens causing disease in humans and other vertebrates, provide the foundation to develop novel strategies for pest control. Here, a series of methods are described that are routinely utilized to determine the functional roles of neuropeptides and other neuronal factors (i.e., biogenic amines) on the excretory system of the mosquito, Aedes aegypti. The Malpighian tubules (MTs), responsible for primary urine formation, can continue functioning for hours when removed from the mosquito, allowing for fluid secretion measurements following hormone treatments. As such, the Ramsay assay is a useful technique to measure secretion rates from isolated MTs. Ion-selective microelectrodes (ISME) can sequentially be used to measure ion concentrations (i.e., Na+ and K+) in the secreted fluid. This assay allows for the measurement of several MTs at a given time, determining the effects of various hormones and drugs. The Scanning Ion-selective Electrode Technique uses ISME to measure voltage representative of ionic activity in the unstirred layer adjacent to the surface of ion transporting organs to determine transepithelial transport of ions in near real time. This method can be used to understand the role of hormones and other regulators on ion absorption or secretion across epithelia. Hindgut contraction assays are also a useful tool to characterize myoactive neuropeptides, that may enhance or reduce the ability of this organ to remove excess fluid and waste. Collectively, these methods provide insight into how the excretory system is regulated in adult mosquitoes. This is important because functional coordination of the excretory organs is crucial in overcoming challenges such as desiccation stress after eclosion and before finding a suitable vertebrate host to obtain a bloodmeal.

This protocol outlines methodologies behind the Ramsay assay, ion-selective microelectrodes, Scanning Ion-selective Electrode Technique (SIET) and in vitro contraction assays, applied to study the adult mosquito excretory system, comprised of the Malpighian tubules and hindgut, to collectively measure ion and fluid secretion rates, contractile activity, and transepithelial ion transport.

研究成年蚊子排泄系统神经肽和其他调节剂的活性

Studies of insect physiology, particularly in those species that are vectors of pathogens causing disease in humans and other vertebrates, provide the ...