这项工作是支持由NIH / NIDCR的格兰特1R01DE019796。

所提出的LEO工作流允许的稳定同位素标记的蛋白质消化和合成肽。这些课程实验（ 图1）是适用于比较和定量蛋白质组学的研究，以及蛋白酶的研究。每个工作流程由两个实验的步骤（ 图2）：A）各自的18 O-稳定的同位素编码的反应（蛋白酶催化肽切割蛋白酶催化羧基氧交换反应，酸催化的羧基氧交换的时间分辨的采样反应）和B）通过质谱法和图形表示的18 O掺入动力学分析。 A.时间过程实验一，古时间过程：蛋白酶催化蛋白水解分裂标签<ol><li> （可选）二硫键的蛋白质（10μM）和肽（250μM）与DTT（终浓度为2.5 mM的减少）在25mM NH 4 HCO 3（两者新鲜制备的）在50℃下孵育30分钟</li><li> （可选）免半胱氨酸用碘乙酰胺烷基化（最终浓度为10毫摩尔）在25mM NH 4 HCO 3在黑暗中在室温下孵育30分钟。 </li><li>根据利益对蛋白酶，蛋白质/肽的解决方案需要清理，以除去残留的缓冲液和烷基化剂。使用PepClean C-18柱（温）的肽清理和VIVASPIN的离心集中器（赛多利斯）蛋白质样品的交换缓冲区。 </li><li>重新溶解/交换在20μl蛋白酶反应缓冲液中的肽/蛋白质（ECE-1：50 mM的MES-KOH，pH值5.5，胰蛋白酶：25 mM的NH 4 HCO 3，pH8.0）中含有1:1（体积/体积）的最终H 2的 18 O（95％，Sigma公司ISOTEC）。 </li><li>蛋白酶到加法之前撤回零时间点样品：将0.5微升的反应混合物和0.5μl的α-氰基-4 - 羟基肉桂酸性基质（10毫克/毫升，50％乙腈，0.1％TFA）。现货样品的Opti-TOF 384的MALDI靶标板（AB SCIEX）留下的溶剂在室温下滴，直到干（约5分钟）。 </li><li>反应溶液中分割成两等份。第一等分试样，将感兴趣的用蛋白酶（ECE-1：75纳米;胰蛋白酶：0.04 nM的）的特定的酶，在室温或所建议的温度孵育。第二份无蛋白酶和培养，将作为对照样本。第一等分试样表示蛋白酶催化的18 O-标记的标准样品，可用于肽和蛋白质鉴定，蛋白水解活性的检测和监测蛋白水解切割反应。第二份是用来显示，18 O公司成立的蛋白酶引起的，因此，既没有标签，也不裂解，预计此示例。 </li><li>按照反应，消除反应装察觉他们的DES第5步的时间间隔cribed下似乎是合适的。上面所述的反应条件是用来监测多达四个天（约24点）的蛋白水解反应。开始取样，每隔5分钟至30分钟，然后发现，每30分钟至2小时，每一个小时，直到8小时，每8个小时，直到4天。裂解产物应该出现在第一个12小时和24小时后的基材应完全水解。延期反应温育时间允许定义稳定的反应产物从反应的中间体，它被进一步处理是离散的。根据研究问题和酶 - 底物对，点状出血时间和孵化温度进行调节，以保证最佳的反应采样。 </li><li>最终的反应时间点之后有斑点或扩展斑点的间隔之间，在MALDI靶板提交MALDI-TOF/TOF MS / MS分析，如下所述（B部分）。 </li></ol> II。古时间过程：Postdigestion标签的蛋白水解总站<ol><li> （可选）二硫键的蛋白质（10μM）和肽（250μM）减少与DTT（终浓度为2.5毫摩尔）在25mM NH 4 HCO 3（包括新鲜制备的）在50℃下30分钟孵育</li><li> （可选）免半胱氨酸用碘乙酰胺烷基化（最终浓度为10毫摩尔）在25mM NH 4 HCO 3在黑暗中在室温下孵育30分钟。 </li><li>根据不同上感兴趣的蛋白酶，蛋白质/肽的解决方案需要清理，以除去残留的缓冲液和烷基化剂。使用PepClean蛋白缓冲交换的肽清理和VIVASPIN的离心浓缩器为C-18柱。 </li><li>重新溶解/交换清洗了肽产品/在20μl蛋白酶反应缓冲液中的蛋白质（ 如胰蛋白酶：25 mM的NH 4 HCO 3，pH为8.0）和消化感兴趣的完成用蛋白酶（ 例如胰蛋白酶：0.04纳米; 37°C，12小时）。 </li><li>清除裂解产物与PepClean C-18离心柱。该步骤可以消除残余的蛋白酶活动。 </li><li>在20μl蛋白酶反应缓冲液重新溶解/交换清理最多肽产品（胰蛋白酶：25mM的NH 4 HCO 3，pH8.0）中含有1:1（体积/体积）的最终H 2 18 O的 </li><li>在加入酶之前撤回零时间点样品：将0.5微升的反应混合物和0.5μl的α-氰基-4 - 羟基肉桂酸的基质（10毫克/毫升，50％乙腈，0.1％TFA）。现货MALDI靶板上的样品和留下的溶剂，在室温下的液滴，直到干燥（约5分钟）。 </li><li>反应溶液中分割成两等份。第一等分试样将与感兴趣的蛋白酶（ 如胰蛋白酶：0.04 nM的）的特定的酶，在室温或所建议的温度孵育。第二份无蛋白酶和培养作为对照。第一等分试样表示标准样品的蛋白酶催化18 O-postdigestion的标签和可用于肽和蛋白质定量。第二份是用来显示，18 O-结合的蛋白酶催化，因此，预计此示例没有标签。 </li><li>按照反应，消除反应装察觉他们根据第7步。）的时间间隔似乎是合适的。例如，我们发现了最初，每5分钟至30分钟，每15分钟后，监测通过胰蛋白酶催化的羧基氧交换反应。 </li><li>在最后的反应时间点有斑点或扩展的斑点间隔之间的的MALDI靶标板被提交到MALDI-TOF/TOF MS / MS分析如下所述（B段）。 </li></ol> III。的ALEO-时间过程：羧基的酸催化的标签<ol><li>孵育个别肽（50纳米），用1:1（体积/体积）18 O浓缩水，在T他的存在或不存在（对照）的0.1％（体积/体积）的最终的三氟乙酸（总体积30μl）。 </li><li>有样品的反应产物通过共48天每天去斑0.5微升等分的混合物与α-氰基-4 - 羟基肉桂酸基质（10毫克/毫升，50％乙腈，0.1％TFA）加入0.5μl直接到MALDI靶板。 </li><li>斑点间隔和对最终反应时间点后斑点提交MALDI靶板MALDI-TOF/TOF MS / MS分析，如下所述部分B之间</li></ol> B. MALDI-TOF/TOF MS / MS数据采集与分析<ol><li>质谱获得4800 MALDI TOF / TOF分析仪（AB SCIEX）。 </li><li>分析前，仪器的校准与质量测量的最大允许误差为±50 ppm和最低数量的六峰匹配的多肽标准（质量标准AB SCIEX TOF / TOF仪器的校准工具包）的混合物。 </li><li> MS谱（质量范围400 - 4,000 米/ z）的购买，使用正离子模式与一个可调的激光强度（3,400 - 3,800，步长50）具有可接受的基峰强度范围为2,000 - 45,000，一式三份。单杆获得子谱，总共有400张/频谱，停止收购条件生效后800分谱（通过或失败）或400分谱合格的验收标准。 MS检测范围的下端低丰度的样品的情况下，或在存在复杂的生物背景应提高到800的m / z。此外，它可能是必要的与样品的清除步骤如前面所述或通过液相色谱分离，以除去盐和其它干扰化合物。 </li><li> MS数据文件（。T2D文件）的出口从4000系列浏览器的数据采集软件，并导入到我们的内部实验室信息系统，该利用MASCOT的蒸馏器软件（矩阵版）的光谱处理和峰值检测。同位素信封解卷积和O-18自动确定使用的算法类似的Mason 等12所描述的，适应我们的9组的掺入比例。另外，如ZoomQuant 13和Viper 14，以及商业套装软件的软件工具， ，随心BioWorks（赛默飞世尔科技）和Mascot Distiller的定量工具箱（矩阵版）解卷积18 O型数据15,16，18 O的注册比率表示的相对贡献的个人肽同位素物种（ 即肽含有16 O 1或18 O，18 O 2）的整个同位素信封。 </li><li>对于每一个时间过程实验，分子量（[M + H] +）可用于所有检测到的肽种类的从相关联的MS数据文件中提取和离散化的值在100 ppm的质量宽度。 </li><li>一至少3 [M + H] +设置为需要填充的垃圾桶。在已知的基板的情况下，滤波后的纸槽列表进行比较到一个列表中的蛋白水解切割产品使用的ExPASy FindPept工具17（ <a href="http://au.expasy.org/tools/findpept.html" target="_blank">http://au.expasy.org/tools/findpept.html</a> ）和一个从底物肽序列预测200 ppm的质量的错误接受性。 </li><li> MS / MS谱获得的所有质量值的裂解产物的FindPept工具，并为BIN值有18个 O-立案法团与预测。 MS / MS数据，4800 MALDI TOF / TOF分析仪在1KV反射器正离子模式下，获得一个固定的激光强度为4200和CID气体在关机模式。 50次收购，在每个子谱随机模式，共40分每点谱（共产生了2000张/点）。 </li><li> MS / MS数据文件（。T2D文件）的出口从4000系列的勘探与RER数据采集软件，并导入我们的内部实验室信息系统，峰值检测和MS / MS峰列表与相应的分级MS的数据。 </li><li> MS / MS肽识别，峰值列表下面的搜索参数对SwissProt数据库使用MASCOT搜索引擎进行搜索，没有酶的特异性，150 ppm的前体离子和0.2的大碎片离子的质量容差。 </li><li>使用Data Explorer软件（AB SCIEX），可以另外进行验证肽鉴定确认的特征18 O-Y系列碎片离子之间的结合模式，舍甫琴科等人所描述的18。 </li></ol> C.制备的频谱时间和18 O公司成立图 频谱时间图：MS每个反应时间点的数据文件（。T2D文件）的出口数据浏览器软件使用的ASCII文件宏观和导入的数据分析和图形软件程序（ 如地OriginLab），并显示为瀑布图（ 图3）。 18 O的注册图：对于每个分级肽裂解产物中提取，个别种肽同位素（16 O，18 O 18 O 2）的相对贡献在所有的反应时间点，对时间作图（ 图4）。

<ol>
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J., Hardt, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acid-catalyzed+oxygen-18+labeling+of+peptides.">Acid-catalyzed oxygen-18 labeling of peptides.</a> Analytical Chemistry. 81, 2804-2809 (2009).</li><li>Bender, M., Stone, R., Dewey, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kinetics+of+Isotopic+Oxygen+Exchange+between+Substituted+Benzoic+Acids+and+Water.">Kinetics of Isotopic Oxygen Exchange between Substituted Benzoic Acids and Water.</a> Journal of the American Chemical Society. 78, 319-321 (1956).</li><li>Kuster, B., Schirle, M., Mallick, P., Aebersold, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Scoring+proteomes+with+proteotypic+peptide+probes.">Scoring proteomes with proteotypic peptide probes.</a> Nature Reviews Molecular Cell Biology. 6, 577-583 (2005).</li></ol>

没有利益冲突的声明。

结合稳定同位素标记和高分辨率质谱在时间分辨的方式，在古时间过程的方法允许产生的肽产品的动态分析。该试剂盒可用于产生稳定的同位素标记的肽进行定量和定性的蛋白质组学研究来评估肽段生成的动力学。此外，古时间过程被设计来评估，根据特定的，生理上有关的条件体外水解途径，可以利用工作流中的内源的蛋白质，肽和蛋白酶，以及合成肽和重组蛋白酶。根据特定的?...

<table border="1"><tbody><tr><td> 材料名称 </td><td> 公司 </td><td> 目录编号 </td></tr><tr><td> PepClean C-18离心柱</td><td>热</td><td> 89870 </td></tr><tr><td>的Opti-TOF 384的MALDI靶标板</td><td> AB SCIEX </td><td> 1016629 </td></tr><tr><td> 4800 MALDI TOF / TOF </td><td> AB SCIEX </td><td></td></tr></tbody></table> 表1。 <table border="1"><tbody><tr><td> 试剂名称 </td><td> 公司 </td><td> 目录编号 </td></tr><tr><td> α-氰基-4 - 羟基肉桂酸</td><td> Sigma Aldrich公司</td><td> 70990-1G-F </td></tr><tr><td>牛血清白蛋白（BSA） </td><td> Sigma Aldrich公司</td><td> A3294-10G </td></tr><tr><td>二硫苏糖醇（DTT）的</td><td&gt; Acros公司</td><td> 16568-0050 </td></tr><tr><td>碘乙酰胺（IAM） </td><td> Sigma Aldrich公司</td><td> 1149-5G </td></tr><tr><td>内皮素转换酶（ECE-1） </td><td> R＆D Systems公司</td><td> 1784-ZN </td></tr><tr><td>胰蛋白酶黄金</td><td> Promega公司</td><td> V5280 </td></tr><tr><td>水18 O，18 O 97％（原子） </td><td> Sigma Aldrich公司</td><td> 329878-1G </td></tr><tr><td>三氟乙酸（TFA） </td><td>热</td><td> 28904 </td></tr><tr><td> AB SCIEX TOF / TOF仪器的校准工具包的质量标准</td><td> AB SCIEX </td><td> 4333604 </td></tr></tbody></table> 表2试剂

protease- and acid-catalyzed labeling workflows employing 18o-enriched water

稳定同位素在生物质谱技术是必不可少的工具。从历史上看，稳定同位素18 O已被广泛用来研究蛋白水解酶1-3的催化机制。随着质谱为基础的蛋白质组学，酶催化成立18 O原子从稳定同位素富集的水已经成为一种流行的方法来定量比较蛋白质表达水平（由Fenselau和姚明，宫城县和4饶5和叶审查等 。6），18 O标记构成一个简单的，低成本的替代化学品（ 如 iTRAQ的，ICAT）和代谢标记技术（ 如 SILAC）。根据使用的蛋白酶的18 O-标记可以导致将多达两个18 O-原子中的C-末端羧基基团的裂解产物3 </sup&gt;。标记反应可以被细分成两个独立的过程，在肽键裂解和羧基氧交换反应8。在我们的古（P rotease 一个的 ssisted的升abelingËmploying 18 O-富氧水）改编酶18 O标记，我们采用50％的18 O富集的水，产生独特的同位素特征。结合高分辨率基质辅助激光解吸电离飞行时间飞行串联质谱（MALDI-TOF/TOF MS / MS），特征同位素信封可以用来识别与高水平的特异性的裂解产品。我们以前使用过的古方法来检测和表征内源性蛋白酶9和监控蛋白水解反应10-11。 ，由于古编码的蛋白裂解反应的本质，实验装置简单，生化ENRIchment步骤裂解产物可以被规避。古方法可以很容易地扩展到（I）课程实验，监测蛋白水解裂解反应的动力学及（ii）分析在复杂的生理条件下的生物样本，分别代表水解。古时间过程的实验有助于确定限速的处理步骤和反应中间体，在复杂的蛋白水解途径反应。此外，古反应，使我们能够识别蛋白水解酶的丝氨酸蛋白酶如胰蛋白酶，能够重新绑定其裂解产物，并促进成立的第二个18 O原子。这种“双标记”酶可用于postdigestion 18 O标记，其中肽是专门标记的羧基氧交换反应。我们的第三个战略扩展标记采用18 O富集的水以外的酶，并使用酸性的pH值条件下，引进18 O稳定同位素标志atures肽。

我们使用动态监控结合成肽蛋白水解酶裂解产生18 O稳定同位素的古时间过程的工作流程。所提出的方法是一种多功能的工具，不同的底物和蛋白酶的组合比较研究蛋白水解处理途径。通过重复采样蛋白水解反应，在反应过程中的，古时间过程的时间分辨实验提供了快照的底物和产物的丰度和处理的详细信息。共发现样本与目标板的酸性基质溶液停止酶的反应和基体结晶，进一步保证样品?...

Watch this Scientific Journal Video about Protease- and Acid-catalyzed Labeling Workflows Employing 18O-enriched Water at JoVE.com

Protease- and Acid-catalyzed Labeling Workflows Employing 18O-enriched Water

稳定同位素标记的工作流程，采用<sup&gt; 18</sup&gt; O-富氧水（LEO-工作流）的多用途工具进行定量和定性的蛋白质组学研究。在蛋白酶辅助（古）的工作流程，<sup&gt; 18</sup&gt; O原子的引入，通过蛋白水解和羧基氧交换反应介导的蛋白酶。在酸催化（ALEO）工作流程中，<sup&gt; 18</sup&gt; O原子在低pH条件下引入羧基氧交换。

蛋白酶和酸催化的贴标工作流程，用人<sup&gt; 18</sup&gt; O-富氧水

Stable isotopes are essential tools in biological mass spectrometry. Historically, 18O-stable isotopes have been extensively used to study the catalytic ...

protease-acid-catalyzed-labeling-workflows-employing-18o-enriched

Research

JoVE Journal

Biology

Protease- and Acid-catalyzed Labeling Workflows Employing 18O-enriched Water

11.9K 次观看.  Boston Biomedical Research Institute. 稳定同位素标记的工作流程，采用 18 O-富氧水（LEO-工作流）的多用途工具进行定量和定性的蛋白质组学研究。在蛋白酶辅助（古）的工作流程， 18 O原子的引入，通过蛋白水解和羧基氧交换反应介导的蛋白酶。在酸催化（ALEO）工作流程中， 18 O原子在低pH条件下引入羧基氧交换。

视频: Protease- and Acid-catalyzed Labeling Workflows Employing 18O-enriched Water

Measuring Exocytosis in Neurons Using FM Labeling

The ability to measure the kinetics of vesicle release can help provide insight into some of the basics of neurotransmission.  Here we used real-time imaging of vesicles labeled with FM dye to monitor the rate of presynaptic vesicle release.  FM4-64 is a red fluorescent amphiphilic styryl dye that embeds into the membranes of synaptic vesicles as endocytosis is stimulated.  Lipophilic interactions cause the dye to greatly increase in fluorescence, thus emitting a bright signal when associated with vesicles and a nominal one when in the extracellular fluid. After a wash step is used to help remove external dye within the plasma membrane, the remaining FM is concentrated within the vesicles and is then expelled when exocytosis is induced by another round of electrical stimulation. The rate of vesicles release is measured from the resulting decrease in fluorescence.  Since FM dye can be applied external and transiently, it is a useful tool for determining rates of exocytosis in neuronal cultures, especially when comparing the rates between transfected synapses and neighboring control boutons.



The ability to measure the kinetics of vesicle release can help provide insight into some of the basics of neurotransmission. Here we used real-time imaging of vesicles labeled with the red fluorescent dye FM 4-64 to measure the rate of presynaptic vesicle release in hippocampal neuronal cultures.

测量中使用FM标签的神经元的胞外分泌

The ability to measure the kinetics of vesicle release can help provide insight into some of the basics of neurotransmission.  Here we used real-time ...

科研

生物学

Morris Water Maze Experiment

The Morris water maze is widely used to study spatial memory and learning. Animals are placed in a pool of water that is colored opaque with powdered non-fat milk or non-toxic tempera paint, where they must swim to a hidden escape platform. Because they are in opaque water, the animals cannot see the platform, and cannot rely on scent to find the escape route. Instead, they must rely on external/extra-maze cues. As the animals become more familiar with the task, they are able to find the platform more quickly. Developed by Richard G. Morris in 1984, this paradigm has become one of the "gold standards" of behavioral neuroscience.

The Morris water maze is a well-accepted tool used to document the involvement of the hippocampus in a behavioral task.

Morris水迷宫实验

The Morris water maze is widely used to study spatial memory and learning. Animals are placed in a pool of water that is colored opaque with powdered ...

Sigma's Non-specific Protease Activity Assay - Casein as a Substrate

Proteases break peptide bonds. In the lab, it is often necessary to measure and/or compare the activity of proteases. Sigma's non-specific protease activity assay may be used as a standardized procedure to determine the activity of proteases, which is what we do during our quality control procedures. In this assay, casein acts as a substrate. When the protease we are testing digests casein, the amino acid tyrosine is liberated along with other amino acids and peptide fragments. Folin and Ciocalteus Phenol, or Folin's reagent primarily reacts with free tyrosine to produce a blue colored chromophore, which is quantifiable and measured as an absorbance value on the spectrophotometer. The more tyrosine that is released from casein, the more the chromophores are generated and the stronger the activity of the protease. Absorbance values generated by the activity of the protease are compared to a standard curve, which is generated by reacting known quantities of tyrosine with the F-C reagent to correlate changes in absorbance with the amount of tyrosine in micromoles. From the standard curve the activity of protease samples can be determined in terms of Units, which is the amount in micromoles of tyrosine equivalents released from casein per minute.
To view this article in Chinese, <a href="http://jove.com/index/details.stp?ID=910">click here</a>

Sigma&#039;s Non-specific Protease Activity Assay - Casein as a Substrate

Proteases break peptide bonds. In the lab, it is often necessary to measure and/or compare the activity of proteases. Sigma's non-specific protease activity assay may be used as a standardized procedure to determine the activity of proteases.

Sigma的非特异性蛋白酶活性测定 - 酪蛋白作为基质

Proteases break peptide bonds. In the lab, it is often necessary to measure and/or compare the activity of proteases.  Sigma's non-specific protease ...

Fluorescent Labeling of Drosophila Heart Structures

The Drosophila melanogaster dorsal vessel, or heart, is a tubular structure comprised of a single layer of contractile cardiomyocytes, pericardial cells that align along each side of the heart wall, supportive alary muscles and, in adults, a layer of ventral longitudinal muscle cells. The contractile fibers house conserved constituents of the muscle cytoarchitecture including densely packed bundles of myofibrils and cytoskeletal/submembranous protein complexes, which interact with homologous components of the extracellular matrix. Here we describe a protocol for the fixation and the fluorescent labeling of particular myocardial elements from the hearts of dissected larvae and semi-intact adult Drosophila. Specifically, we demonstrate the labeling of sarcomeric F-actin and of &alpha;-actinin in larval hearts. Additionally, we perform labeling of F-actin and &alpha;-actinin in myosin-GFP expressing adult flies and of &alpha;-actinin and pericardin, a type IV extracellular matrix collagen, in wild type adult hearts. Particular attention is given to a mounting strategy for semi-intact adult hearts that minimizes handling and optimizes the opportunity for maintaining the integrity of the cardiac tubes and the associated tissues. These preparations are suitable for imaging via fluorescent and confocal microscopy. Overall, this procedure allows for careful and detailed analysis of the structural characteristics of the heart from a powerful genetically tractable model system.

Fluorescent Labeling of Drosophila Heart Structures

Here we describe a basic protocol for fluorescent labeling of different elements of heart tubes from larva and adult Drosophila melanogaster. These specimens are well-suited for imaging via fluorescent or confocal microscopy. This technique permits detailed structural analysis of the features of the hearts from a powerful model organism.

荧光标记的果蝇心结构

The Drosophila melanogaster dorsal vessel, or heart, is a tubular structure comprised of a single layer of contractile cardiomyocytes, pericardial cells ...

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 ...

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. ...

Mouse Islet of Langerhans Isolation using a Combination of Purified Collagenase and Neutral Protease

The interrogation of beta cell gene expression and function in vitro has squarely shifted over the years from the study of rodent tumorigenic cell lines to the study of isolated rodent islets. Primary islets offer the distinct advantage that they more faithfully reflect the biology of intracellular signaling pathways and secretory responses. Whereas the method of islet isolation using tissue dissociating enzyme (TDE) preparations has been well established in many laboratories1-4, variations in the consistency of islet yield and quality from any given rodent strain limit the extent and feasibility of primary islet studies. These variations often occur as a result of the crude partially purified TDEs used in the islet isolation procedure; TDEs frequently exhibit lot-to-lot variations in activity and often require adjustments to the dose of enzyme used. A small number of reports have used purified TDEs for rodent cell isolations5, 6, but the practice is not widespread despite the routine use and advantages of purified TDEs for human islet isolations. In collaboration with VitaCyte, LLC (Indianapolis, IN), we developed a modified mouse islet isolation protocol based on that described by Gotoh7, 8, in which the TDEs are perfused directly into the pancreatic duct of mice, followed by crude tissue fractionation through a Histopaque gradient9, and isolation of purified islets. A significant difference in our protocol is the use of purified collagenase (CIzyme MA) and neutral protease (CIzyme BP) combination. The collagenase was characterized by the use of a6 fluorescence collagen degrading activity (CDA) assay that utilized fluorescently labeled soluble calf skin fibrils as substrate6. This substrate is more predictive of the kinetics of collagen degradation in the tissue matrix because it relies on native collagen as the substrate. The protease was characterized with a sensitive fluorescent kinetic assay10. Utilizing these improved assays along with more traditional biochemical analysis enable the TDE to be manufactured more consistently, leading to improved performance consistency between lots. The protocol described in here was optimized for maximal islet yield and optimal islet morphology using C57BL/6 mice. During the development of this protocol, several combinations of collagenase and neutral proteases were evaluated at different concentrations, and the final ratio of collagenase:neutral protease of 35:10 represents enzyme performance comparable to Sigma Type XI. Because significant variability in average islet yields from different strains of rats and mice have been reported, additional modifications of the TDE composition should be made to improve the yield and quality of islets recovered from different species and strains.

A detailed description of mouse islet isolation is described using the technique of in situ pancreatic ductal cannulation and perfusion of a combination of purified collagenase and neutral protease.

小鼠胰岛的朗格汉斯的分离纯化的胶原酶和中性蛋白酶结合使用

The interrogation of beta cell gene expression and function in vitro has squarely shifted over the years from the study of rodent tumorigenic cell lines ...

Determining Membrane Protein Topology Using Fluorescence Protease Protection (FPP)

The correct topology and orientation of integral membrane proteins are essential for their proper function, yet such information has not been established for many membrane proteins. A simple technique called fluorescence protease protection (FPP) is presented, which permits the determination of membrane protein topology in living cells. This technique has numerous advantages over other methods for determining protein topology, in that it does not require the availability of multiple antibodies against various domains of the membrane protein, does not require large amounts of protein, and can be performed on living cells. The FPP method employs the spatially confined actions of proteases on the degradation of green fluorescent protein (GFP) tagged membrane proteins to determine their membrane topology and orientation. This simple approach is applicable to a wide variety of cell types, and can be used to determine membrane protein orientation in various subcellular organelles such as the mitochondria, Golgi, endoplasmic reticulum and components of the endosomal/recycling system. Membrane proteins, tagged on either the N-termini or C-termini with a GFP fusion, are expressed in a cell of interest, which is subject to selective permeabilization using the detergent digitonin. Digitonin has the ability to permeabilize the plasma membrane, while leaving intracellular organelles intact. GFP moieties exposed to the cytosol can be selectively degraded through the application of protease, whereas GFP moieties present in the lumen of organelles are protected from the protease and remain intact. The FPP assay is straightforward, and results can be obtained rapidly.

Determining Membrane Protein Topology Using Fluorescence Protease Protection FPP

Here, we present a protocol to determine the orientation and topology of integral membrane proteins in living cells. This simple protocol relies on selective protease sensitivity of chimeras between the protein of interest and GFP.

确定膜蛋白拓扑使用荧光蛋白酶保护（FPP）

The correct topology and orientation of integral membrane proteins are essential for their proper function, yet such information has not been established ...

使用机器视觉方法的 TEM 工作流程、结果分析和数据管理

A Machine-Vision Approach to Transmission Electron Microscopy Workflows, Results Analysis and Data Management

Transmission electron microscopy (TEM) enables users to study materials at their fundamental, atomic scale. Complex experiments routinely generate thousands of images with numerous parameters that require time-consuming and complicated analysis. AXON synchronicity is a machine-vision synchronization (MVS) software solution designed to address the pain points inherent to TEM studies. Once installed on the microscope, it enables the continuous synchronization of images and metadata generated by the microscope, detector, and in situ systems during an experiment. This connectivity enables the application of machine-vision algorithms that apply a combination of spatial, beam, and digital corrections to center and track a region of interest within the field of view and provide immediate image stabilization. In addition to the substantial improvement in resolution afforded by such stabilization, metadata synchronization enables the application of computational and image analysis algorithms that calculate&#8239;variables between images. This calculated metadata can be used to analyze trends or identify key areas of interest within a dataset, leading to new insights and the development of more sophisticated machine-vision capabilities in the future. One such module that builds on this calculated metadata is dose calibration and management. The dose module provides state-of-the-art calibration, tracking, and management of both the electron fluence (e-/&#197;2&#183;s-1) and cumulative dose (e-/&#197;2) that is delivered to specific areas of the sample on a pixel-by-pixel basis. This enables a comprehensive overview of the interaction between the electron beam and the sample. Experiment analysis is streamlined through a dedicated analysis software in which datasets consisting of images and corresponding metadata are easily visualized, sorted, filtered, and exported. Combined, these tools facilitate efficient collaborations and experimental analysis, encourage data mining and enhance the microscopy experience.

作者聚焦：用于透射电子显微镜工作流程、结果分析和数据管理的机器视觉方法  

Here, we present a protocol to utilize machine-vision software to stabilize dynamic processes during TEM imaging, while concurrently indexing multiple streams of metadata to each image into a navigable timeline. We demonstrate how this platform enables automated calibration and mapping of the electron dose over the course of an experiment.