我们承认梅利莎Grunlan SAM的形成有益的讨论。这项研究是支持的，一部分由罗伯特A ·韦尔奇基金会（1585年）。

试剂的制备准备管道的缓冲液（80毫米的管道，1MM氯化镁2，为1mM EGTA，用KOH调节pH至6.8）。分装和储存葡萄糖氧化酶，过氧化氢 ​​酶和葡萄糖在-70 ° C和紫杉醇在-20 ° C根据现有的协议。1,2分装和存储微管蛋白在-70 ° C，根据供应商包括的指示。 图案的金微电极使用光刻<ol><li>切成1厘米，1.5厘米，用抄写员或晶圆切割机基板的硅晶片。他们有足够的丙酮，在烧杯中，以支付他们，超声5分钟的清洁基板。而不让基板干燥，新鲜丙酮冲洗，然后立即在异丙醇和干燥使用氮气冲洗。 </li><li>工房金微电极的清洁基板上使用的标准，积极音光刻或电子束光刻，根据所需的功能的大小3当设计模式的几何形状，保持直径100微米以下的每个电极图案覆盖，以确保聚乙二醇的SAM（在下一节中描述）。包括接触片（〜300毫米2）从300到500毫米1毫米的电极垫（图1）连接线的格局定位。基板中间附近的平版印刷的图案，让周围的组装流室的格局空间。 <img border="0" src="/files/ftp_upload/1390/1390fig1thumb.jpg" alt="图1" /> 图1。样品的几何形状。请<a href="https://www.jove.com/files/ftp_upload/1390/1390fig1.jpg">点击这里</a>看到图1的放大版本。 </li><li>在光阻层的模式发展后，从头开始在抵制从接触垫的使用镊子（图1）基板的边缘线。从头金属沉积后，将使电极与基板边缘之间的电接触。沉积室，最好是有两个来源，是用来沉积的金属层。其次为6纳米的金，不破坏真空的情况下，沉积的Cr 2纳米（粘连）所形成的电极。箱内配备石英晶体微量天平，可用于测量薄膜的厚度在沉积过程中。 </li></ol>准备的SAM <ol><li>预热烤箱在通风良好的区域为75 ° C准备一个250毫升的玻璃烧杯加入20 mL甲苯的硅烷化解决方案。然后加入1毫升的PEG -硅烷（即5％V / V）甲苯，用塑料吸管。 PEG硅烷吹打高达5至10倍，然后用30秒的吸管搅拌混合。 </li><li>烧杯图案的一面朝上放置在图案的样品，确保它们完全淹没在PEG硅烷的甲苯溶液，并均匀地分布在烧杯底部间隔。烧杯放置在烤箱和烘烤18至21小时，在75 ° C。虽然SAM正在形成，准备counterelectrode（见下文）和组装中的其余步骤所需的任何额外的元件。 </li><li>在烘箱中18-21小时后，甲苯挥发一种粘稠的PEG -硅烷残渣留在图案的样本。加入20 mL甲苯烧杯中，浸泡1-2分钟的样品。从烧杯中取出样品，用甲苯，然后异丙醇冲洗，最后用氮气干燥。此时，样品应显得干净和SAM层应该是肉眼看不见的。地空导弹准备这两天使用，储存在阴凉干燥条件下（25℃，湿度适中）。 SAM在阴天或不均匀斑驳陆离残留涂层表面上形成的不成功结果。 </li></ol>编造的counterelectrode 在同一沉积室设置用于微电极沉积，沉积1 nm的铬，4纳米金上一个22 × 50毫米的1号或0号玻璃盖玻片counterelectrode。 counterelectrode应接近透明的浅灰色或粉红色的色调。商店在培养皿counterelectrode涂层朝上，以保持清洁。 到MTS的聚合微管蛋白<ol><li>解冻未标记的微管蛋白和罗丹明微管蛋白，并立即贴上标签，无标签，以获得明亮的微管在1:2的比例结合。混合移液器向上和向下几次。在恒温水浴20-40分钟的微管蛋白聚合，在37 ° C。多边贸易体制的长度将是更大的聚合时间较长。虽然微管蛋白聚合，准备加入到含有微量管道的缓冲区管的紫杉醇20毫米的一个管道紫杉醇的缓冲区。紫杉醇混合移液器向上和向下几次震荡，然后放入加热浴管在37 ° C。聚合反应后，1 / 100稀释的MTS在暖管道紫杉醇缓冲区和盾由轻。在这种方式准备的MTS将拉长随着时间的推移和捆绑，它可以减少被进一步稀释。 </li><li>准备30毫升冷管道的缓冲区（0-4℃），5毫升50％2 -巯基乙醇，5毫升5毫升的葡萄糖氧化酶，过氧化氢 ​​酶，和5 mL葡萄糖结合了10倍的氧气清除混合物（OS） 。葡萄糖应该是最后添加。混合吹打后加入每个组件向上和向下的3-5倍。操作系统的混合物置于冰上;将保持约2小时有效。 </li><li>验证吨聚合是成功的，多边贸易体系的稳定1X操作系统组合的准备微管成像的荧光激发下几分钟。成像，这种方式准备了MTS，一定要使用适合罗丹明荧光激发/发射滤​​波器组合。确定进一步稀释1 / 10到1 / 100使用1X操作系统组合的管道缓冲区和成像的MTS个人细丝成像的最佳工作稀释。 </li><li>准备一个流室，夹两片双面胶带分开放置2-3毫米的一个大（22 × 50毫米）和小（22 × 22毫米）的玻璃盖玻片。磁带很容易切成条状，放在载玻片上，并用刀片切割。引入流使用微量的细胞MTS的几个微升。对于高放大倍率的成像，长工作距离的目标可能需要成像的上表面流室。从盖玻片/样品表面的距离上表面流室是由磁带的厚度，应60-100毫米。 </li></ol>图案MT的使用电泳<ol><li>准备通过将整个图案，SAM涂层样品，电极之间的连接路径到磁带上（图2）垂直的条两个细条双面胶带电泳室。与金属的一面触摸磁带等counterelectrode放在双面胶带，约3毫米（图2）样品出挑的counterelectrode的边缘。切几个15厘米的绝缘铜线的长度和钢带两端的1厘米的保温。将电线外露的金基板面积的使用银粉漆的小降，同时注意避免使电气接触与counterelectrode（这可以用万用表检查）。将第二线counterelectrode使用银粉漆。银漆连接线，可以进一步固定用胶带样品。 <img src="/files/ftp_upload/1390/1390fig2thumb.jpg" border="0" alt="图2" /> 图2。流倒置荧光显微镜下的细胞大会。请<a href="https://www.jove.com/files/ftp_upload/1390/1390fig2.jpg">点击这里</a>看大图图2。 </li><li>使用管道缓冲区1X终浓度与操作系统相结合，在所需的稀释介绍MTS。在20倍的放大倍率图像和定位在施加电压的硅晶片上的图案。 （可选），流室，可密封使用指甲油，以防止流体流动和流室的平面MT迁移。 </li><li>申请高达1伏的电压，并观察MTS的迁移。如果可逆捕获所需的MTS，可用于低电压（下降约0.5 V的），但是这需要密封，以防止横向漂移流室。指甲油密封流室的开放结束。在约0.7 V以上的电压，可能会看到一些吨粘附上的图案，虽然这将是微弱的。在电压约0.9 V以上，吨迁移将更快，粘连会更强。约1.2伏以上的诱导电解（气泡的形成）和破坏的微电极的可能性大大增加。迁移可以跟踪通过调整显微镜的焦点垂直平面。如果正确执行，MTS将在电极表面的本地化。 </li></ol>

<ol>
	<li>Rai-Choudhury, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Handbook of Microlithography, Micromachining, and Microfabrication: Microlithography. , (1997).</li></ol>

The authors have nothing to disclose.

MTS的迁移是很容易的可视化使用，因为它们的亮度和低背景荧光的荧光显微镜。方法一般适用于生物分子的图案，因为它仅需要诱导的生物分子进行适当的调整缓冲液pH值的净电荷。电渗是避免在此设置，因为有没有收取的表面，如毛细管电泳中使用的硅墙壁。 这种方法的若干修改是可能的。密封流室的两端是至关重要的观察，因为从开完的蒸发导致散装流体流动和不良吨?...

从VWR购买1号高硼硅玻璃盖玻片（22 × 50毫米）。抛光电子级p型&lt;111&gt;硅片由Addison工程公司购买了高纯度的银粉漆是从结构探头，公司（目录＃5001 AB）获得。绝缘细针距铜线（〜0.08毫米（0.003）直径）可从电子供应公司。

antifouling self-assembled monolayers on microelectrodes for patterning biomolecules

我们目前与金微电极的硅衬底上形成一聚乙二醇（PEG）的三甲氧基硅烷自组装单层膜（SAM）的一个过程。 PEG - SAM是形成一个单一的装配步骤，防止在硅和金表面的生物污染。 SAM是用来与标准，积极音光刻图案的大衣微电极。微管作为一个例子，我们应用诱导电泳迁移到一个可逆的方式SAM涂层电极直流电压。甲流室是用于成像的电泳迁移和微管的图案<em&gt;原位</em&gt;使用epifluorescence显微镜。这种方法一般适用于生物分子的图案，因为它采用电泳固定靶标分子，因此不需要特定的分子间的相互作用。此外，它避免了试图模式自组装单分子直接使用平版印刷技术时所遇到的问题。允许这种方法用于模式生物分子在纳米电子束光刻技术的兼容性。

Watch this Scientific Journal Video about Antifouling Self-assembled Monolayers on Microelectrodes for Patterning Biomolecules at JoVE.com

Antifouling Self-assembled Monolayers on Microelectrodes for Patterning Biomolecules

我们目前与金微电极的硅衬底上形成一个聚乙二醇（PEG）自组装单层膜（PEG - SAM）的一个过程。 PEG - SAM是形成了一个步骤，并防止在硅和金表面的生物污染。电泳是用于图案的生物分子到纳米级。

防污图案生物分子自组装膜的微电极为

We present a procedure for forming a poly(ethylene glycol) (PEG) trimethoxysilane self-assembled monolayer (SAM) on a silicon substrate with gold ...

antifouling-self-assembled-monolayers-on-microelectrodes-for

Research

JoVE Journal

Biology

11.4K 次观看.  Texas A&M University (TAMU). 我们目前与金微电极的硅衬底上形成一个聚乙二醇（PEG）自组装单层膜（PEG - SAM）的一个过程。 PEG - SAM是形成了一个步骤，并防止在硅和金表面的生物污染。电泳是用于图案的生物分子到纳米级。

视频: Antifouling Self-assembled Monolayers on Microelectrodes for Patterning Biomolecules

Patterning Cells on Optically Transparent Indium Tin Oxide Electrodes

The ability to exercise precise spatial and temporal control over cell-surface interactions is an important prerequisite to the assembly of multi-cellular constructs serving as in vitro mimics of native tissues.  In this study, photolithography and wet etching techniques were used to fabricate individually addressable indium tin oxide (ITO) electrodes on glass substrates.  The glass substrates containing ITO microelectrodes were modified with poly(ethylene glycol) (PEG) silane to make them protein and cell resistive.  Presence of insulating PEG molecules on the electrode surface was verified by cyclic voltammetry employing potassium ferricyanide as a redox reporter molecule.  Importantly, the application of reductive potential caused desorption of the PEG layer, resulting in regeneration of the conductive electrode surface and appearance of typical ferricyanide redox peaks.    Application of reductive potential also corresponded to switching of ITO electrode properties from cell non-adhesive to cell-adhesive. Electrochemical stripping of PEG-silane layer from ITO microelectrodes allowed for cell adhesion to take place in a spatially defined fashion, with cellular patterns corresponding closely to electrode patterns.  Micropatterning of several cell types was demonstrated on these substrates.   In the future, the control of the biointerfacial properties afforded by this method will allow to engineer cellular microenvironments through the assembly of three or more cell types into a precise geometric configuration on an optically transparent substrate.

Non-fouling PEG silane monolayer was desorbed from individually addressable ITO electrodes on glass by application of a reductive potential. Electrochemical stripping of PEG-silane layer from ITO microelectrodes allowed for cell adhesion to take place in a spatially defined fashion, with cellular patterns corresponding closely to electrode patterns.

在光学透明的铟锡氧化物电极的图案细胞

The ability to exercise precise spatial and temporal control over cell-surface interactions is an important prerequisite to the assembly of multi-cellular ...

科研

生物学

Patterning of Embryonic Stem Cells Using the Bio Flip Chip

Cell-cell interactions consisting of diffusible signaling and cell-cell contact (juxtacrine signaling) are important in numerous biological processes such as tumor growth, stem cell differentiation, and stem cell self-renewal. A number of methods currently exist to modulate cell signaling in vitro. One method of modulating the total amount of diffusible signaling is to vary the cell seeding density during culture. Due to the random nature of cell seeding, this results in considerable variation in the actual cell-cell spacing and amount of cell-cell contact, and cannot prescribe the local environment. A more specific approach for modulating cell signaling is to use molecular inhibitors or genetic approaches to knock down specific signaling proteins, but both of these methods are best suited to manipulating small numbers of molecules. Here, we demonstrate a new approach to modulating cell-cell signaling that modulates the local environment of a cluster of cells by placing different numbers of cells at desired locations on a substrate. This method provides a complementary way to control the local diffusible and juxtacrine signaling between cells. Our method makes use of the Bio Flip Chip (BFC), a microfabricated silicone chip containing hundreds-to-thousands of microwells, each sized to hold either a single cell or small numbers of cells. We load the chip with cells simply by pipetting them onto the array of wells and washing unloaded cells off the array. The chip is then flipped onto a substrate, whereby the cells fall out of the wells and onto the substrate, maintaining their patterning. After the cells have attached, the chip can be removed (or left on). This approach to cell patterning is unique in that it: 1) doesn't alter the chemistry of the substrate, thus allowing cells to proliferate and migrate; 2) allows patterning onto any substrate, including tissue-culture polystyrene, glass, matrigel, and even feeder cell layers; and 3) is compatible with traditional microcontact printing, allowing the creation of extracellular matrix islands with cells placed inside those islands. In this video, we demonstrate the patterning of mouse embryonic stem cells onto tissue-culture polystyrene using the BFC.

We demonstrate a simple method for placing cells at desired locations on a substrate. This method patterns cells by flipping a silicone chip containing microwells filled with cells onto the substrate. This method provides a new way to modulate diffusible and juxtacrine signaling between cells.

图案的使用生物倒装芯片的胚胎干细胞

Cell-cell interactions consisting of diffusible signaling and cell-cell contact (juxtacrine signaling) are important in numerous biological processes such ...

Automated System for Single Molecule Fluorescence Measurements of Surface-immobilized Biomolecules

Fluorescence Resonance Energy Transfer (FRET) microscopy has been widely used to study the structure and dynamics of molecules of biological interest, such as nucleic acids and proteins. Single molecule FRET (sm-FRET) measurements on immobilized molecules permit long observations of the system -effectively until both dyes photobleach- resulting in time-traces that report on biomolecular dynamics with a broad range of timescales from milliseconds to minutes. To facilitate the acquisition of large number of traces for statistical analyses, the process must be automated and the sample environment should be tightly controlled over the entire measurement time (~12 hours). This is accomplished using an automated scanning confocal microscope that allows the interrogation of thousands of single molecules overnight, and a microfluidic cell that permits the controlled exchange of buffer, with restricted oxygen content and maintains a constant temperature throughout the entire measuring period. Here we show how to assemble the microfluidic device and how to activate its surface for DNA immobilization. Then we explain how to prepare a buffer to maximize the photostability and lifetime of the fluorophores. Finally, we show the steps involved in preparing the setup for the automated acquisition of time-resolved single molecule FRET traces of DNA molecules.

In this article we describe how we obtain FRET traces from individual DNA molecules immobilized to a surface using an automated scanning confocal microscope.

表面固定生物分子的单分子荧光检测的自动化系统

Fluorescence Resonance Energy Transfer (FRET) microscopy has been widely used to study the structure and dynamics of molecules of biological interest, ...

Single Cell Transcriptional Profiling of Adult Mouse Cardiomyocytes

While numerous studies have examined gene expression changes from homogenates of heart tissue, this prevents studying the inherent stochastic variation between cells within a tissue. Isolation of pure cardiomyocyte populations through a collagenase perfusion of mouse hearts facilitates the generation of single cell microarrays for whole transcriptome gene expression, or qPCR of specific targets using nanofluidic arrays. We describe here a procedure to examine single cell gene expression profiles of cardiomyocytes isolated from the heart. This paradigm allows for the evaluation of metrics of interest which are not reliant on the mean (for example variance between cells within a tissue) which is not possible when using conventional whole tissue workflows for the evaluation of gene expression (Figure 1). We have achieved robust amplification of the single cell transcriptome yielding micrograms of double stranded cDNA that facilitates the use of microarrays on individual cells. In the procedure we describe the use of NimbleGen arrays which were selected for their ease of use and ability to customize their design. Alternatively, a reverse transcriptase - specific target amplification (RT-STA) reaction, allows for qPCR of hundreds of targets by nanofluidic PCR. Using either of these approaches, it is possible to examine the variability of expression between cells, as well as examining expression profiles of rare cell types from within a tissue. Overall, the single cell gene expression approach allows for the generation of data that can potentially identify idiosyncratic expression profiles that are typically averaged out when examining expression of millions of cells from typical homogenates generated from whole tissues.

Single cell expression profiling allows the detailed gene expression analysis of individual cells. We describe methods for the isolation of cardiomyocytes, and preparing the resulting lysates for either whole transcriptome microarray or qPCR of specific targets.

成年小鼠心肌细胞的单细胞转录谱

While numerous studies have examined gene expression changes from homogenates of heart tissue, this prevents studying the inherent stochastic variation ...

Protein WISDOM: A Workbench for In silico De novo Design of BioMolecules

The aim of de novo protein design is to find the amino acid sequences that will fold into a desired 3-dimensional structure with improvements in specific properties, such as binding affinity, agonist or antagonist behavior, or stability, relative to the native sequence. Protein design lies at the center of current advances drug design and discovery. Not only does protein design provide predictions for potentially useful drug targets, but it also enhances our understanding of the protein folding process and protein-protein interactions. Experimental methods such as directed evolution have shown success in protein design. However, such methods are restricted by the limited sequence space that can be searched tractably. In contrast, computational design strategies allow for the screening of a much larger set of sequences covering a wide variety of properties and functionality. We have developed a range of computational de novo protein design methods capable of tackling several important areas of protein design. These include the design of monomeric proteins for increased stability and complexes for increased binding affinity.
To disseminate these methods for broader use we present Protein WISDOM (<a href="http://www.proteinwisdom.org" target="_blank">http://www.proteinwisdom.org</a>), a tool that provides automated methods for a variety of protein design problems. Structural templates are submitted to initialize the design process. The first stage of design is an optimization sequence selection stage that aims at improving stability through minimization of potential energy in the sequence space. Selected sequences are then run through a fold specificity stage and a binding affinity stage. A rank-ordered list of the sequences for each step of the process, along with relevant designed structures, provides the user with a comprehensive quantitative assessment of the design. Here we provide the details of each design method, as well as several notable experimental successes attained through the use of the methods.

Protein WISDOM: A Workbench for In silico De novo Design of BioMolecules

We developed computational de novo protein design methods capable of tackling several important areas of protein design. To disseminate these methods we present Protein WISDOM, an online tool for protein design (<a href="http://www.proteinwisdom.org" target="_blank">http://www.proteinwisdom.org</a>). Starting from a structural template, design of monomeric proteins for increased stability and complexes for increased binding affinity can be performed.

蛋白质智慧：一个工作台<em&gt;硅片</em&gt;<em&gt;从头</em&gt;生物分子设计

The aim of de novo protein design is to find the amino acid sequences that will fold into a desired 3-dimensional structure with improvements in specific ...

Internalization and Observation of Fluorescent Biomolecules in Living Microorganisms via Electroporation

The ability to study biomolecules in vivo is crucial for understanding their function in a biological context. One powerful approach involves fusing molecules of interest to fluorescent proteins such as GFP to study their expression, localization and function. However, GFP and its derivatives are significantly larger and less photostable than organic fluorophores generally used for in vitro experiments, and this can limit the scope of investigation. 
We recently introduced a straightforward, versatile and high-throughput method based on electroporation, allowing the internalization of biomolecules labeled with organic fluorophores into living microorganisms. Here we describe how to use electroporation to internalize labeled DNA fragments or proteins into Escherichia coli and Saccharomyces cerevisi&#230;, how to quantify the number of internalized molecules using fluorescence microscopy, and how to quantify the viability of electroporated cells. Data can be acquired at the single-cell or single-molecule level using fluorescence or FRET. The possibility of internalizing non-labeled molecules that trigger a physiological observable response in vivo is also presented. Finally, strategies of optimization of the protocol for specific biological systems are discussed.

Studies of biomolecules in vivo are crucial for understanding molecular function in a biological context. Here we describe a novel method allowing the internalization of fluorescent biomolecules, such as DNA or proteins, into living microorganisms. Analysis of in vivo data recorded by fluorescence microscopy is also presented and discussed.

通过电穿孔内在和生物分子荧光观察生活中的微生物

The ability to study biomolecules in vivo is crucial for understanding their function in a biological context. One powerful approach involves fusing ...

Measurement and Analysis of Extracellular Acid Production to Determine Glycolytic Rate

Extracellular measurement of oxygen consumption and acid production is a simple and powerful way to monitor rates of respiration and glycolysis1. Both mitochondrial (respiration) and non-mitochondrial (other redox) reactions consume oxygen, but these reactions can be easily distinguished by chemical inhibition of mitochondrial respiration. However, while mitochondrial oxygen consumption is an unambiguous and direct measurement of respiration rate2, the same is not true for extracellular acid production and its relationship to glycolytic rate 3-6. Extracellular acid produced by cells is derived from both lactate, produced by anaerobic glycolysis, and CO2, produced in the citric acid cycle during respiration. For glycolysis, the conversion of glucose to lactate- + H+ and the export of products into the assay medium is the source of glycolytic acidification. For respiration, the export of CO2, hydration to H2CO3 and dissociation to HCO3- + H+ is the source of respiratory acidification. The proportions of glycolytic and respiratory acidification depend on the experimental conditions, including cell type and substrate(s) provided, and can range from nearly 100% glycolytic acidification to nearly 100% respiratory acidification 6. Here, we demonstrate the data collection and calculation methods needed to determine respiratory and glycolytic contributions to total extracellular acidification by whole cells in culture using C2C12 myoblast cells as a model.

Glycolysis is a defining metabolic marker in multiple biological systems. Monitoring glycolysis by measuring the extracellular flux of H+ is common, but requires correction to be quantitative and unambiguous. Here, we demonstrate how to gather and correct extracellular flux data to distinguish between respiratory and glycolytic sources of extracellular acidification.

测量外产酸和分析，以确定糖酵解率

Extracellular measurement of oxygen consumption and acid production is a simple and powerful way to monitor rates of respiration and glycolysis1. Both ...

Isolation of Specific Genomic Regions and Identification of Associated Molecules by enChIP

The identification of molecules associated with specific genomic regions of interest is required to understand the mechanisms of regulation of the functions of these regions. To enable the non-biased identification of molecules interacting with a specific genomic region of interest, we recently developed the engineered DNA-binding molecule-mediated chromatin immunoprecipitation (enChIP) technique. Here, we describe how to use enChIP to isolate specific genomic regions and identify the associated proteins and RNAs. First, a genomic region of interest is tagged with a transcription activator-like (TAL) protein or a clustered regularly interspaced short palindromic repeats (CRISPR) complex consisting of a catalytically inactive form of Cas9 and a guide RNA. Subsequently, the chromatin is crosslinked and fragmented by sonication. The tagged locus is then immunoprecipitated and the crosslinking is reversed. Finally, the proteins or RNAs that are associated with the isolated chromatin are subjected to mass spectrometric or RNA sequencing analyses, respectively. This approach allows the successful identification of proteins and RNAs associated with a genomic region of interest.

The identification of molecules associated with specific genomic regions of interest is required to understand the mechanisms of regulation of the functions of these regions. This protocol describes procedures to perform engineered DNA-binding molecule-mediated chromatin imunoprecipitation (enChIP) for identification of proteins and RNAs associated with a specific genomic region.

具体的基因组区域及相关分子鉴定enChIP隔离

The identification of molecules associated with specific genomic regions of interest is required to understand the mechanisms of regulation of the ...

Quantification of Site-specific Protein Lysine Acetylation and Succinylation Stoichiometry Using Data-independent Acquisition Mass Spectrometry

Post-translational modification (PTM) of protein lysine residues by N&#400;-acylation induces structural changes that can dynamically regulate protein functions, for example, by changing enzymatic activity or by mediating interactions. Precise quantification of site-specific protein acylation occupancy, or stoichiometry, is essential for understanding the functional consequences of both global low-level stoichiometry and individual high-level acylation stoichiometry of specific lysine residues. Other groups have reported measurement of lysine acetylation stoichiometry by comparing the ratio of peptide precursor isotopes from endogenous, natural abundance acylation and exogenous, heavy isotope-labeled acylation introduced after quantitative chemical acetylation of proteins using stable isotope-labeled acetic anhydride. This protocol describes an optimized approach featuring several improvements, including: (1) increased chemical acylation efficiency, (2) the ability to measure protein succinylation in addition to acetylation, and (3) improved quantitative accuracy due to reduced interferences using fragment ion quantification from data-independent acquisitions (DIA) instead of precursor ion signal from data-dependent acquisition (DDA). The use of extracted peak areas from fragment ions for quantification also uniquely enables differentiation of site-level acylation stoichiometry from proteolytic peptides containing more than one lysine residue, which is not possible using precursor ion signals for quantification. Data visualization in Skyline, an open source quantitative proteomics environment, allows for convenient data inspection and review. Together, this workflow offers unbiased, precise, and accurate quantification of site-specific lysine acetylation and succinylation occupancy of an entire proteome, which may reveal and prioritize biologically relevant acylation sites.

Here, we present unbiased quantification of site-specific protein acetylation and/or succinylation occupancy (stoichiometry) of an entire proteome through a ratiometric analysis of endogenous modifications to modifications introduced after quantitative chemical acylation using stable isotope-labeled anhydrides. In combination with sensitive data-independent acquisition mass spectrometry, accurate site occupancy measurements are obtained.

用数据无关采集质谱法定量测定点特异蛋白赖氨酸乙酰化和琥珀酰化化学计量学

Post-translational modification (PTM) of protein lysine residues by N&#400;-acylation induces structural changes that can dynamically regulate protein ...

SA-&#946;-Galactosidase-Based Screening Assay for the Identification of Senotherapeutic Drugs

Cell senescence is one of the hallmarks of aging known to negatively influence a healthy lifespan. Drugs able to kill senescent cells specifically in cell culture, termed senolytics, can reduce the senescent cell burden in vivo and extend healthspan. Multiple classes of senolytics have been identified to date including HSP90 inhibitors, Bcl-2 family inhibitors, piperlongumine, a FOXO4 inhibitory peptide and the combination of Dasatinib/Quercetin. Detection of SA-&#946;-Gal at an increased lysosomal pH is one of the best characterized markers for the detection of senescent cells. Live cell measurements of senescence-associated &#946;-galactosidase (SA-&#946;-Gal) activity using the fluorescent substrate C12FDG in combination with the determination of the total cell number using a DNA intercalating Hoechst dye opens the possibility to screen for senotherapeutic drugs that either reduce overall SA-&#946;-Gal activity by killing of senescent cells (senolytics) or by suppressing SA-&#946;-Gal and other phenotypes of senescent cells (senomorphics). Use of a high content fluorescent image acquisition and analysis platform allows for the rapid, high throughput screening of drug libraries for effects on SA-&#946;-Gal, cell morphology and cell number.

SA-&amp;#946;-Galactosidase-Based Screening Assay for the Identification of Senotherapeutic Drugs

Cellular senescence is the key factor in the development of chronic age-related pathologies. Identification of therapeutics that target senescent cells show promise for extending healthy aging. Here, we present a novel assay to screen for the identification of senotherapeutics based on measurement of senescence associated &#946;-Galactosidase activity in single cells.

SA-β-加拉基沙酶筛查检测,用于识别治疗药物

Cell senescence is one of the hallmarks of aging known to negatively influence a healthy lifespan. Drugs able to kill senescent cells specifically in cell ...