我们感谢自然科学和工程研究理事会（NSERC）和加拿大癌症协会提供财政支持。 SCCS的感谢NSERC和VNL感谢安大略研究生奖学金研究生奖学金（OGS）计划。 ARW的一个加拿大研究主席感谢“儿童权利公约”。

第1部分：设备制造<ol><li>食人鱼的解决方案中的清洁玻璃基板（3:1浓硫酸：30％双氧水）。让食人鱼频繁搅拌10分钟的解决方案的基板。 </li><li>在去离子（DI）水冲洗后干燥与N 2气体的基板，铬沉积（厚度为250 nm）的电子束室里面放置的基板。 </li><li>脱水铬涂层基板，在异丙醇冲洗，然后在5分钟的热板烘烤在115 ° C。 </li><li>通过旋涂（30秒，3000转），干与六甲基二硅氮烷（HMDS）的基板和总理。自旋大衣希普利S1811光刻胶（使用相同的参数）。 </li><li>预烘基板上的热板（100℃，2分钟），然后模式由暴露在紫外线（UV）照射5通过光罩小号光致抗蚀剂。 </li><li>希普利MF 321 3分钟和DI水冲洗开发商开发的紫外线暴露基板。后烤热板在100 ° C，持续1分钟。 </li><li>蚀刻暴露铬铬蚀刻液浸泡30秒在去离子水冲洗，然后在10分钟AZ300T脱衣舞的浸泡，以去除剩余的光致抗蚀剂。在DI水冲洗和干燥与N 2气体。 </li><li>存款2-5微米的聚对二甲苯C（绝缘聚合物）通过化学气相沉积到基板图案铬轴承。存款50纳米聚四氟乙烯的AF旋涂在2000转的解决方案（1％Fluorinert WT / WT在FC - 40）（使表面疏水）60秒后烤热板（160℃，10分钟）。 </li><li>要形成顶板，大衣联合国图案的铟锡氧化物（ITO）玻璃基板50纳米聚四氟乙烯的AF，如上。 </li><li>后烤热板（160℃，10分钟）都基板。 </li></ol>第2部分：设备设置及自动化<ol><li>在“两板”配置操作的数字微流体装置，1液滴之间的底部基板（图案电极）和顶部基板（一个连续的，透明电极，通常形成从ITO）的位置。 </li><li>要设置设备，取出一个用手术刀轻轻地刮取底部基板的接触片的聚合物涂层。夫妻暴露的底部基板焊盘与40针连接器（图1a）。 </li><li>电源的计算机上运行的LabView（美国国家仪器公司，美国德克萨斯州奥斯汀​​），一个自制的控制箱（具有高电压继电器阵列是由一个国家仪器DaqPad信号控制），函数发生器，放大器。计算机/控制箱，方便用户超过100 V RMS / 18 kHz的信号通过40针连接器的设备应用程序的控制。 </li><li>组装定位于边缘的底部基板双面胶带两件（总厚度为140微米）的设备，并完成与unpatterned ITO幻灯片（顶板）。 </li><li>要初始化的控制系统，运行LabVIEW校准程序（内部书面）校准的反馈控制。 </li><li>装入适当水库（见下面的第3条，）的样品和试剂，并附上顶下基板（聚四氟乙烯涂层的一面朝下）。将接地连接器顶端的基板。 </li><li>在LabVIEW中加载的驱动代码（内部书面）和执行程序启动液滴驱动。 </li></ol>第3部分：样品和试剂的制备<ol><li>准备工作缓冲区（WB）：100毫米TrisHCl（pH值7.8）和0.08％聚醚F127 W / V。 </li><li>解散蛋白（S）在世界银行和吸管样品水库1（R - 1的）设备上的分析，如图1b所示。 </li><li>准备到R - 2 DI水的沉淀，20％三氯乙酸（TCA）和吸管。 </li><li>准备洗涤缓冲液，70/30氯仿/乙腈（ACN）和吸管到R - 4。 </li><li>准备到R - 5 resolubilizing缓冲区，100毫米碳酸氢铵（pH值8.0）和0.08％W / V，聚醚F127和吸管。 </li><li>还原剂， 三 （2 -羧乙基）膦（TCEP），溶解在世界银行和吸管在10毫米到R - 6。 </li><li>烷化剂，碘乙酰胺（IAM），溶解在世界银行和吸管在12毫米到R - 7。 </li><li>世界银行在一个平等的总蛋白样品浓度（见3.1以上）和吸管TO1 / 5到R - 8的浓度溶解胰蛋白酶。 </li></ol>第4部分：数字微流体样品处理<ol><li>自动执行下面的过程是由内部编写的LabVIEW代码。从水库配发的水滴的体积是指由电极的尺寸（在这种情况下，配发的水滴〜600nL）。 </li><li>分配从R - 1的含蛋白质样品液滴和液滴的沉淀，从R - 2（图2，第1帧）。合并两个水滴，并允许合并的液滴孵育5分钟，导致PR降水在表面上oteins（图2，帧2-3）。驱动上清液从沉淀的蛋白质废物水库，为R - ​​3（图2，第4帧）。 </li><li>免除从R - 4三种洗涤缓冲液的液滴，并推动整个沉淀蛋白质的浪费水库，为R - ​​3（图2，第5帧）。 </li><li>允许沉淀，干燥，免除从R - 5 resolubilizing缓冲液滴的蛋白质。允许缓冲区孵育20分钟，直到沉淀溶解（图2，帧6）。 </li><li>免除从R - 6液滴的还原剂和合并与样品液滴。混合驱动在一个圆形图案，它横跨6个电极相结合的液滴。允许液滴在湿盒（图3，帧1-2）为在室温下1小时孵育。 </li><li>分送一个液滴的R - 7烷基化剂和混合（4.5），与样品液滴合并。允许液滴孵育15 min，室温避光，在湿盒（图3，帧2-3）。 </li><li>液滴的胰蛋白酶，免除从R - 8和混合（4.5），与样品液滴合并。允许液滴孵育3小时37 °中的一个热点板块的湿盒（图3，帧3-4）彗星。 </li></ol>第5部分：处理后的样品制备<ol><li>卸下顶部基板和淬火消化反应，2.5％三氟乙酸在水中加入0.6毫升。 </li><li>净化淬火后的反应产物，根据制造商的指示使用（Millipore公司，比尔里卡，马萨诸塞州）C18 ZipTips。 </li><li> DI水稀释纯化样品终体积为100 mL。 </li></ol>第6部分：质谱<ol><li>评估交配串联质谱仪在纳米高效液相色谱法处理样品。高效液相色谱系统用在这里是从Eksigent技术和装备是一个陷阱列（3毫米直径的。C18的珠子，150微米ID x 2.5厘米）和一个反相分离柱（直径3微米。C18的珠子，75微米内径x 15厘米）。这里使用的是热尔科技的LTQ质谱仪。 </li><li>在移动，包括5％乙腈水阶段，加载到5μL/min陷阱列2μL样品。在0.5μL/min反相列使用线性渐变，从20％乙腈超过25分钟至80％的乙腈样品分开。再过10分钟，继续运行在80％乙腈。 </li><li>洗脱列串联质谱分析频段。在这里的工作报告，洗脱液进入质谱仪通过nanoelectrospray发射器（20微米收窄至10微米的ID编号）从NewObjective采样。 </li><li>使用SEQUEST等数据库检索程序，确定样品中的蛋白质。在这项工作中，我们使用的版本SEQUEST Bioworks V3.01（热尔科技）成立，并确定蛋白质的概率分数小于1.0E - 03（相当于99.9％的置信区间），并在序列覆盖至少30％（图4）。 </li></ol><img src="/files/ftp_upload/1603/1603fig1a.jpg" align="Figure 1a" /> <img src="/files/ftp_upload/1603/1603fig1b.jpg" align="Figure 1b" /> 图1（a）一个图片一个二甲基甲酰胺装置配备40针连接器自动化液滴驱动。 （二）设备原理图描绘了一个蛋白质组学workup所需样品和试剂的定位。 <img src="/files/ftp_upload/1603/1603fig2.jpg" align="Figure 2" /> 从电影图2。帧描绘在20％TCA（沉淀）和70/30％氯仿/乙腈（冲洗液）自动提取和纯化的BSA。在第6帧，沉淀蛋白质溶解在100毫米碳酸氢铵液滴。 <img src="/files/ftp_upload/1603/1603fig3.jpg" align="Figure 3" /> 图3说明顺序还原，烷基化，消化resolubilized蛋白液滴从一个电影帧。在这个数字中，所用试剂为清晰起见，染料着色，在实践中，所用试剂均为不着色。 <img src="/files/ftp_upload/1603/1603fig4.jpg" align="Figure 4" /> 图4。MS色谱数字微流体处理的牛血清白蛋白的样品。 25个不同的肽（99.9％的置信区间），相应序列覆盖了44％。

<ol>
	<li>Abdelgawad, M., Wheeler, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Digital+Revolution:+A+New+Paradigm+for+Microfluidics.">The Digital Revolution: A New Paradigm for Microfluidics.</a> Adv. Mat. 21, 920-925 (2009).</li><li>Jebrail, M., Wheeler, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Digital+Microfluidic+Method+for+Protein+Extraction+by+Precipitation.">A Digital Microfluidic Method for Protein Extraction by Precipitation.</a> Anal. Chem. 81, 330-335 (2009).</li><li>Luk, V. N., Wheeler, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Digital+Microfluidic+Approach+to+Proteomic+Sample+Processing.">A Digital Microfluidic Approach to Proteomic Sample Processing.</a> Anal. Chem. 81, 4524-4530 (2009).</li></ol>

缺乏规范的样品处理和加工蛋白质组学是该领域的主要的限制。此外，传统的宏观样品处理涉及多个容器和解决方案转移，从而导致样品的损失和污染。这些问题的潜在解决方案是要形成综合系统为依托数字微流体1甲酰胺（DMF）的样品处理。 2同样，二甲基甲酰胺被证明是一个集成与整合多级的解决方案相处理（还原，烷基化和消化）兼容在以往的工作，DMF被证明是有效去除不必...

digital microfluidics for automated proteomic processing

临床蛋白质组学研究已成为一个重要的新的学科，有前途的生物标志物，将有助于疾病的早期诊断和预后的发现。临床蛋白质组学方法虽然千差万别，一个共同的特点是：（一）需要极其庞杂的液体（如血清，全血等）和（二）广泛的生化处理，分析前中提取蛋白质。在这里，我们报告一个新的数字微流体（DMF）为基础的方法相结合用于临床蛋白质组学的几个处理步骤。这包括蛋白的提取，resolubilization，还原，烷基化和酶消化。数字微流体是一个微型的流体处理技术，在一个开放的表面上操纵纳升，微升大小的水滴。液滴涂绝缘层的电极阵列上的定位 - 当一个电势应用到液滴，收费积累的介质两侧。收费作为静电处理，可以用来控制液滴的位置，并通过偏置系列电极序列，水滴可以被免除，移动，合并，组合，和表面上的分裂。因此，DMF是一种自然适合进行快速，连续，多步，小型化自动生化分析。这意味着超过常规方法（依靠手动移液器或机器人）的一个显着的进步，并有可能成为一个有用的临床蛋白质组学的新工具。 MAIS研究杰布拉伊尔，利德北鹿，和史蒂夫消委会施同样对这项工作。 塞尔吉奥LS弗莱雷的当前地址是在费城科学大学位于南43街600，费城，PA 19104。

Watch this Scientific Journal Video about Digital Microfluidics for Automated Proteomic Processing at JoVE.com

Digital Microfluidics for Automated Proteomic Processing

数字微流体操纵离散液滴（NL - ML）的特点是技术，电气领域的应用电极阵列。它是非常适合进行快速，连续化，小型化自动生化分析。在这里，我们报告的一个平台，能够自动化几个蛋白质组学的处理步骤。

数字微流体进行自动蛋白质组学处理

Clinical proteomics has emerged as an important new discipline, promising the discovery of biomarkers that will be useful for early diagnosis and ...

digital-microfluidics-for-automated-proteomic-processing

Research

JoVE Journal

Biology

12.4K 次观看.  University of Toronto. 数字微流体操纵离散液滴（NL - ML）的特点是技术，电气领域的应用电极阵列。它是非常适合进行快速，连续化，小型化自动生化分析。在这里，我们报告的一个平台，能够自动化几个蛋白质组学的处理步骤。

视频: Digital Microfluidics for Automated Proteomic Processing

Chemotactic Response of Marine Micro-Organisms to Micro-Scale Nutrient Layers

The degree to which planktonic microbes can exploit microscale resource patches will have considerable implications for oceanic trophodynamics and biogeochemical flux. However, to take advantage of nutrient patches in the ocean, swimming microbes must overcome the influences of physical forces including molecular diffusion and turbulent shear, which will limit the availability of patches and the ability of bacteria to locate them. Until recently, methodological limitations have precluded direct examinations of microbial behaviour within patchy habitats and realistic small-scale flow conditions. Hence, much of our current knowledge regarding microbial behaviour in the ocean has been procured from theoretical predictions. To obtain new information on microbial foraging behaviour in the ocean we have applied soft lithographic fabrication techniques to develop 2 microfluidic devices, which we have used to create (i) microscale nutrient patches with dimensions and diffusive characteristics relevant to oceanic processes and (ii) microscale vortices, with shear rates corresponding to those expected in the ocean. These microfluidic devices have permitted a first direct examination of microbial swimming and chemotactic behaviour within a heterogeneous and dynamic seascape. The combined use of epifluorescence and phase contrast microscopy allow direct examinations of the physical dimensions and diffusive characteristics of nutrient patches, while observing the population-level aggregative response, in addition to the swimming behaviour of individual microbes. These experiments have revealed that some species of phytoplankton, heterotrophic bacteria and phagotrophic protists are adept at locating and exploiting diffusing microscale resource patches within very short time frames. We have also shown that up to moderate shear rates, marine bacteria are able to  fight the flow  and swim through their environment at their own accord. However, beyond a threshold high shear level, bacteria are aligned in the shear flow and are less capable of swimming without disturbance from the flow. Microfluidics represents a novel and inexpensive approach for studying aquatic microbial ecology, and due to its suitability for accurately creating realistic flow fields and substrate gradients at the microscale, is ideally applicable to examinations of microbial behaviour at the smallest scales of interaction.  We therefore suggest that microfluidics represents a valuable tool for obtaining a better understanding of the ecology of microorganisms in the ocean.

The fabrication of microfluidic channels and their implementation in experiments for studying the chemotactic foraging behaviour of marine microbes within a patchy nutrient seascape and the swimming behaviour of bacteria within shear flow are described.

海洋微生物的趋化反应的微观尺度的营养层

The degree to which planktonic microbes can exploit microscale resource patches will have considerable implications for oceanic trophodynamics and ...

科研

生物学

Studies of Bacterial Chemotaxis Using Microfluidics - Interview

使用微流体的细菌的趋化作用的研究 - 访

Applying Microfluidics to Electrophysiology

Microfluidics can be integrated with standard electrophysiology techniques to allow new experimental modalities.  Specifically, the motivation for the microfluidic brain slice device is discussed including how the device docks to standard perfusion chambers and the technique of passive pumping which is used to deliver boluses of neuromodulators to the brain slice.  By simplifying the device design, we are able to achieve a practical solution to the current unmet electrophysiology need of applying multiple neuromodulators across multiple regions of the brain slice.  This is achieved by substituting the standard coverglass substrate of the perfusion chamber with a thin microfluidic device bonded to the coverglass substrate.  This was then attached to the perfusion chamber and small holes connect the open-well of the perfusion chamber to the microfluidic channels buried within the microfluidic substrate.  These microfluidic channels are interfaced with ports drilled into the edge of the perfusion chamber to access and deliver stimulants.  This project represents how the field of microfluidics is transitioning away from proof-of concept device demonstrations and into practical solutions for unmet experimental and clinical needs.

应用微流体电

Microfluidics can be integrated with standard electrophysiology techniques to allow new experimental modalities.  Specifically, the motivation for the ...

Electrophoretic Separation of Proteins

Electrophoresis is used to separate complex mixtures of proteins (e.g., from cells, subcellular fractions, column fractions, or immunoprecipitates), to investigate subunit compositions, and to verify homogeneity of protein samples. It can also serve to purify proteins for use in further applications. In polyacrylamide gel electrophoresis, proteins migrate in response to an electrical field through pores in a polyacrylamide gel matrix; pore size decreases with increasing acrylamide concentration. The combination of pore size and protein charge, size, and shape determines the migration rate of the protein. In this unit, the standard Laemmli method is described for discontinuous gel electrophoresis under denaturing conditions, i.e., in the presence of sodium dodecyl sulfate (SDS). 

In this video, we demonstrate a method for electrophoretic separation of proteins using poly-acrylimide gel electrophoresis (PAGE).

电泳分离蛋白质

Electrophoresis is used to separate complex mixtures of proteins (e.g., from cells, subcellular fractions, column fractions, or immunoprecipitates), to ...

Immunoblot Analysis

Immunoblotting (western blotting) is a rapid and sensitive assay for the detection and characterization of proteins that works by exploiting the specificity inherent in antigen-antibody recognition. It involves the solubilization and electrophoretic separation of proteins, glycoproteins, or lipopolysaccharides by gel electrophoresis, followed by quantitative transfer and irreversible binding to nitrocellulose, PVDF, or nylon. The immunoblotting technique has been useful in identifying specific antigens recognized by polyclonal or monoclonal antibodies and is highly sensitive (1 ng of antigen can be detected). This unit provides protocols for protein separation, blotting proteins onto membranes, immunoprobing, and visualization using chromogenic or chemiluminescent substrates.

Immunoblotting (western blotting) is a rapid and sensitive assay for the detection and characterization of proteins that works by exploiting the specificity inherent in antigen-antibody recognition. This video provides protocols for protein separation, blotting proteins onto membranes, immunoprobing, and visualization using chromogenic or chemiluminescent substrates.

免疫印迹分析

Immunoblotting (western blotting) is a rapid and sensitive assay for the detection and characterization of proteins that works by exploiting the ...

Staining Proteins in Gels

Following separation by electrophoretic methods, proteins in a gel can be detected by several staining methods. This unit describes protocols for detecting proteins by four popular methods. Coomassie blue staining is an easy and rapid method. Silver staining, while more time consuming, is considerably more sensitive and can thus be used to detect smaller amounts of protein. Fluorescent staining is a popular alternative to traditional staining procedures, mainly because it is more sensitive than Coomassie staining, and is often as sensitive as silver staining. Staining of proteins with SYPRO Orange and SYPRO Ruby are also demonstrated here.

Following separation by electrophoretic methods, proteins in a gel can be detected by several staining methods. Staining of proteins with Coomassie Blue, Silver Staining, SYPRO Orange, SYPRO Ruby are demonstrated in this video.

在凝胶染色蛋白质

Following separation by electrophoretic methods, proteins in a gel can be detected by several staining methods. This unit describes protocols for ...

Toxin Induction and Protein Extraction from Fusarium spp. Cultures for Proteomic Studies

Fusaria are filamentous fungi able to produce different toxins. Fusarium mycotoxins such as deoxynivalenol, nivalenol, T2, zearelenone, fusaric acid, moniliformin, etc... have adverse effects on both human and animal health and some are considered as pathogenicity factors. Proteomic studies showed to be effective for deciphering toxin production mechanisms (Taylor et al., 2008) as well as for identifying potential pathogenic factors (Paper et al., 2007, Houterman et al., 2007) in Fusaria. It becomes therefore fundamental to establish reliable methods for comparing between proteomic studies in order to rely on true differences found in protein expression among experiments, strains and laboratories. The procedure that will be described should contribute to an increased level of standardization of proteomic procedures by two ways. The filmed protocol is used to increase the level of details that can be described precisely. Moreover, the availability of standardized procedures to process biological replicates should guarantee a higher robustness of data, taking into account also the human factor within the technical reproducibility of the extraction procedure.
The protocol described requires 16 days for its completion: fourteen days for cultures and two days for protein extraction (figure 1). 
Briefly, Fusarium strains are grown on solid media for 4 days; they are then manually fragmented and transferred into a modified toxin inducing media (Jiao et al., 2008) for 10 days. Mycelium is collected by filtration through a Miracloth layer. Grinding is performed in a cold chamber. Different operators performed extraction replicates (n=3) in order to take into account the bias due to technical variations (figure 2). Extraction was based on a SDS/DTT buffer as described in Taylor et al. (2008) with slight modifications. Total protein extraction required a precipitation process of the proteins using Aceton/TCA/DTT buffer overnight and Acetone /DTT washing (figure 3a,3b). Proteins were finally resolubilized in the protein-labelling buffer and quantified. Results of the extraction were visualized on a 1D gel (Figure 4, SDS-PAGE), before proceeding to 2D gels (IEF/SDS-PAGE). The same procedure can be applied for proteomic analyses on other growing media and other filamentous fungi (Miles et al., 2007).

Toxin Induction and Protein Extraction from Fusarium spp. Cultures for Proteomic Studies

Protein extraction for proteomic analyses in fungal species requires high levels of standardization to be accomplished according with the minimum information about a proteomic experiment (MIAPE) guidelines. We present a video-protocol that includes a procedure for minimizing experimental bias during toxin induction and protein extraction from Fusarium spp.

毒素诱导和蛋白质提取镰刀 SPP。</em蛋白质组学研究>文化

Fusaria are filamentous fungi able to produce different toxins. Fusarium mycotoxins such as deoxynivalenol, nivalenol, T2, zearelenone, fusaric acid, ...

Evisceration of Mouse Vitreous and Retina for Proteomic Analyses

While the mouse retina has emerged as an important genetic model for inherited retinal disease, the mouse vitreous remains to be explored. The vitreous is a highly aqueous extracellular matrix overlying the retina where intraocular as well as extraocular proteins accumulate during disease.1-3 Abnormal interactions between vitreous and retina underlie several diseases such as retinal detachment, proliferative diabetic retinopathy, uveitis, and proliferative vitreoretinopathy.1,4 The relative mouse vitreous volume is significantly smaller than the human vitreous (Figure 1), since the mouse lens occupies nearly 75% of its eye.5 This has made biochemical studies of mouse vitreous challenging. In this video article, we present a technique to dissect and isolate the mouse vitreous from the retina, which will allow use of transgenic mouse models to more clearly define the role of this extracellular matrix in the development of vitreoretinal diseases.

The dissection technique illustrates evisceration of the vitreous, retina, and lens from the mouse eye, separation by centrifugation, and characterization with protein assays.

眼内容剜除小鼠玻璃体和视网膜蛋白质组分析

While the mouse retina has emerged as an important genetic model for inherited retinal disease, the mouse vitreous remains to be explored. The vitreous is ...

Metabolic Labeling and Membrane Fractionation for Comparative Proteomic Analysis of Arabidopsis thaliana Suspension Cell Cultures

Plasma membrane microdomains are features based on the physical properties of the lipid and sterol environment and have particular roles in signaling processes. Extracting sterol-enriched membrane microdomains from plant cells for proteomic analysis is a difficult task mainly due to multiple preparation steps and sources for contaminations from other cellular compartments. The plasma membrane constitutes only about 5-20% of all the membranes in a plant cell, and therefore isolation of highly purified plasma membrane fraction is challenging. A frequently used method involves aqueous two-phase partitioning in polyethylene glycol and dextran, which yields plasma membrane vesicles with a purity of 95% 1. Sterol-rich membrane microdomains within the plasma membrane are insoluble upon treatment with cold nonionic detergents at alkaline pH. This detergent-resistant membrane fraction can be separated from the bulk plasma membrane by ultracentrifugation in a sucrose gradient 2. Subsequently, proteins can be extracted from the low density band of the sucrose gradient by methanol/chloroform precipitation. Extracted protein will then be trypsin digested, desalted and finally analyzed by LC-MS/MS. Our extraction protocol for sterol-rich microdomains is optimized for the preparation of clean detergent-resistant membrane fractions from Arabidopsis thaliana cell cultures.
We use full metabolic labeling of Arabidopsis thaliana suspension cell cultures with K15NO3 as the only nitrogen source for quantitative comparative proteomic studies following biological treatment of interest 3. By mixing equal ratios of labeled and unlabeled cell cultures for joint protein extraction the influence of preparation steps on final quantitative result is kept at a minimum. Also loss of material during extraction will affect both control and treatment samples in the same way, and therefore the ratio of light and heave peptide will remain constant. In the proposed method either labeled or unlabeled cell culture undergoes a biological treatment, while the other serves as control 4.

Metabolic Labeling and Membrane Fractionation for Comparative Proteomic Analysis of Arabidopsis thaliana Suspension Cell Cultures

Here we describe a robust method for the fractionation of plant plasma membranes into detergent resistant and detergent soluble membranes based on a mixture of unlabeled and in vivo fully 15N labeled Arabidopsis thaliana cell cultures. The procedure is applied for comparative proteomic studies to understand signaling processes.

代谢标记和膜分馏的比较蛋白质组学分析<em&gt;拟南芥</em&gt;悬浮细胞培养

Plasma membrane microdomains are features based on the physical properties of the lipid and sterol environment and have particular roles in signaling ...

Magnetic-Activated Cell Sorting Strategies to Isolate and Purify Synovial Fluid-Derived Mesenchymal Stem Cells from a Rabbit Model

Mesenchymal stem cells (MSCs) are the main cell source for cell-based therapy. MSCs from articular cavity synovial fluid could potentially be used for cartilage tissue engineering. MSCs from synovial fluid (SF-MSCs) have been considered promising candidates for articular regeneration, and their potential therapeutic benefit has made them an important research topic of late. SF-MSCs from the knee cavity of the New Zealand white rabbit can be employed as an optimized translational model to assess human regenerative medicine. By means of CD90-based magnetic activated cell sorting (MACS) technologies, this protocol successfully obtains rabbit SF-MSCs (rbSF-MSCs) from this rabbit model and further fully demonstrates the MSC phenotype of these cells by inducing them to differentiate to osteoblasts, adipocytes, and chondrocytes. Therefore, this approach can be applied in cell biology research and tissue engineering using simple equipment and procedures.

This article presents a simple and economic protocol for the straightforward isolation and purification of mesenchymal stem cells from New Zealand white rabbit synovial fluid.

从兔模型中分离纯化滑液源性间充质干细胞的磁活化细胞分选策略

Mesenchymal stem cells (MSCs) are the main cell source for cell-based therapy. MSCs from articular cavity synovial fluid could potentially be used for ...