这项工作是支持由美国国立卫生研究院拨款RO1 DC006497，RO1 NS057289，PO1 ES016738（张到Z）;加州再生医学赠款，RS1 - 00331 - 1研究所和RL1 - 00682 - 1（张到Z）;美国帕金森病协会（Z. Zhang和黏Choo的）和迈克尔J.福克斯帕金森病研究基金会（Z.张）。

在培养细胞中检测蛋白的泛素化<ol><li>与表达的蛋白（抗原表位标签的版本）泛的质粒转染培养细胞。 </li><li>请完整的细胞裂解液（2％十二烷基硫酸钠，氯化钠150毫米，10毫米的Tris - HCl，pH值8.0）2MM钒酸钠，50毫米氟化钠，和蛋白酶抑制剂。 </li><li>裂解细胞，每盘100μL细胞裂解液（6厘米菜）。如果使用一个更大的盘，相应地调整音量。仔细旋流菜让裂解液覆盖整个区域的细胞生长。 </li><li>与细胞刮刀收集细胞和细胞裂解液转移到1.5 mL Eppendorf管。管放置到一个热点板块，立即煮沸10分钟。 </li><li>剪切细胞与超声设备。 </li><li>加入900ul稀释缓冲液（10毫米的Tris - HCl，pH值8.0，氯化钠150毫米，2毫米EDTA，1％的Triton）。在4 ° C孵育30-60分钟与旋转样品。 </li><li>在20,000 30分钟XG的旋转稀释样品。导致上清转移到一个新的Eppendorf管。要小心不要去打扰颗粒。 </li><li>测定蛋白浓度。 </li><li>准备对一个或G琼脂糖珠共轭抗体在兼容的缓冲液（50％浆）的目标蛋白的蛋白质。切窄的一端和一个P - 200枪头14-20树脂液转移到500-1,500微克准备的免疫细胞裂解液。细胞转染效率高，500微克就足够了。对于细胞的转染效率低，可能需要更多的蛋白质。 </li><li>在4 ° C过夜旋转培养的细胞裂解液珠的混合物。 </li><li>离心5分钟在5000 XG珠。吸出上清液。洗净树脂洗涤缓冲液（10毫米的Tris - HCl，pH值8.0，1 M氯化钠，1毫米EDTA，1％NP - 40）的两倍。 </li><li>旋转作最后一次为30秒20,000 XG珠。吸残留的洗涤液和2X SDS上样缓冲液煮沸的树脂。 </li><li>装载到SDS - PAGE凝胶免疫分析的样品。 </li><li>检测泛素和相应的抗体与靶蛋白。对于免疫印迹，我们一般先检测泛素。膜会被用来检测蛋白质的沉淀。 </li></ol> 在体外泛素化实验检测靶蛋白通过泛素化<ol><li>设为5X泛素化缓冲液（100毫米的Tris - HCl，pH值7.5，25毫米氯化镁，数码地面电视的2.5毫米，10毫米ATP）。储存在-20 ° C长达6个月的小等分。有些协议也添加到缓冲区ATP再生1,2磷酸肌酸和肌酸磷酸激酶。 </li><li>对于每一个反应，准备的混合物，包含以下内容： <table border="1"><tbody><tr><td> 8μL </td><td> 5X泛素缓冲区</td></tr><tr><td> 250吴</td><td>泛素E1 </td></tr><tr><td> 500吴</td><td>泛素E2 </td></tr><tr><td> 0.5微克</td><td>泛</td></tr><tr><td> 0.5微克</td><td>蛋白质的利益</td></tr><tr><td></td><td>水至40μL总体积</td></tr></tbody></table>作为对照，无论是E1，E2或泛的情况下，准备在类似的反应。 </li><li>混合物在37℃，1小时或更长的时间。 </li><li>加入SDS - PAGE样品缓冲液停止反应，煮沸10分钟的样品。 </li><li>加载到SDS - PAGE电泳，免疫印迹分析的样品。 </li><li>检测泛素和相应的抗体与靶蛋白。 </li></ol>

<ol>
	<li>Xiong, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PINK1,+and+DJ-1+form+a+ubiquitin+E3+ligase+complex+promoting+unfolded+protein+degradation.">PINK1, and DJ-1 form a ubiquitin E3 ligase complex promoting unfolded protein degradation.</a> J Clin Invest. 119, 650-660 (2009).</li><li>Xirodimas, D. P., Saville, M. K., Bourdon, J. C., Hay, R. T., Lane, D. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mdm2-mediated+NEDD8+conjugation+of+p53+inhibits+its+transcriptional+activity.">Mdm2-mediated NEDD8 conjugation of p53 inhibits its transcriptional activity.</a> Cell. 118, 83-97 (2004).</li><li>Didier, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNF5,+a+RING+finger+protein+that+regulates+cell+motility+by+targeting+paxillin+ubiquitination+and+altered+localization.">RNF5, a RING finger protein that regulates cell motility by targeting paxillin ubiquitination and altered localization.</a> Mol Cell Biol. 23, 5331-5345 (2003).</li><li>Laney, J. D., Hochsetrasser, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Unit+14.5+Analysis+of+Protein+Ubiquitination.">Unit 14.5 Analysis of Protein Ubiquitination.</a> Current Protocols in Protein Science. , 14.5.1-14.5.11 (2002).</li></ol>

The authors have nothing to disclose.

在此演示中，我们首先描述的步骤，允许检测的兴趣培养的哺乳动物细胞中的蛋白质的泛素修饰。为了检测蛋白质的利益，不会对非共价键相互作用的蛋白质，泛素化，特别是，我们用一个严格的条件下裂解细胞，免疫沉淀和洗涤 1,3 。泛素化，反泛素免疫印迹在这样一个苛刻的条件的目标蛋白的免疫检测，因此可能是特定目标蛋白质。为了防止在实验过程中的蛋白质deubiquitination， 如...

detection of protein ubiquitination

泛素化，多肽靶蛋白的泛素共价结合，是一个重要的三种酶进行转录后修饰。它们包括泛素激活酶E1，泛素结合酶E2和泛素连接酶E3。不同于以E1和E2，E3泛素连接酶显示底物特异性。另一方面，众多deubiquitylating酶处理polyubiquitinated蛋白质的作用。泛素蛋白的稳定性，细胞定位及生物活性的变化可能导致。在参与泛素/ deubiquitination途径或系统功能改变泛的基因突变与许多不同的人类疾病，如癌症，神经退行性疾病和代谢性疾病，各类。改变或正常靶蛋白的泛素化的检测可能会提供一个更好地了解这些疾病的发病机制。在这里，我们描述了协议检测培养细胞中的蛋白质的泛素<em&gt;在体内</em&gt;和试管<em&gt;在体外</em&gt;。这些协议也用于检测其他泛素样小分子修饰，如sumolyation和neddylation。

Watch this Scientific Journal Video about Detection of Protein Ubiquitination at JoVE.com

Detection of Protein Ubiquitination

泛素化是一个关键的三种酶进行转录后修饰。在此修改有关的基因突变与许多不同的人类疾病。在这里，我们描述了协议检测培养细胞中的蛋白质的泛素<em&gt;在体内</em&gt;和试管<em&gt;在体外</em&gt;。

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 ...

Research

JoVE Journal

Biology

42.6K 次观看.  The Sanford Burnham Institute for Medical Research. 泛素化是一个关键的三种酶进行转录后修饰。在此修改有关的基因突变与许多不同的人类疾病。在这里，我们描述了协议检测培养细胞中的蛋白质的泛素在体内和试管在体外。

视频: Detection of Protein Ubiquitination

Batch Immunostaining for Large-Scale Protein Detection in the Whole Monkey Brain

Immunohistochemistry (IHC) is one of the most widely used laboratory techniques for the detection of target proteins in situ. Questions concerning the expression pattern of a target protein across the entire brain are relatively easy to answer when using IHC in small brains, such as those of rodents. However, answering the same questions in large and convoluted brains, such as those of primates presents a number of challenges. Here we present a systematic approach for immunodetection of target proteins in an adult monkey brain. This approach relies on the tissue embedding and sectioning methodology of NeuroScience Associates (NSA) as well as tools developed specifically for batch-staining of free-floating sections. It results in uniform staining of a set of sections which, at a particular interval, represents the entire brain. The resulting stained sections can be subjected to a wide variety of analytical procedures in order to measure protein levels, the population of neurons expressing a certain protein.

Large-scale immunodetection of target proteins across the entire primate brain is possible by employing novel tissue embedding and sectioning methods combined with the use of creative apparatus for batch staining of multiple free-floating sections at a given time.

在整个猴脑进行大规模蛋白质检测的批次免疫染色

Immunohistochemistry (IHC) is one of the most widely used laboratory techniques for the detection of target proteins in situ. Questions concerning the ...

科研

生物学

Staining of Proteins in Gels with Coomassie G-250 without Organic Solvent and Acetic Acid

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents (Methanol, Ethanol or 2-Propanol) and acetic acid are used for fixation, staining and destaining of proteins in a gel after SDS-PAGE. To speed up the procedure, heating the staining solution in the microwave oven for a short time is frequently used. This usually results in evaporation of toxic or hazardous Methanol, Ethanol or 2-Propanol and a strong smell of acetic acid in the lab which should be avoided due to safety considerations. In a protocol originally published in two patent applications by E.M. Wondrak (US2001046709 (A1), US6319720 (B1)), an alternative composition of the staining solution is described in which no organic solvent or acid is used. The CBB is dissolved in bidistilled water (60-80mg of CBB G-250 per liter) and 35 mM HCl is added as the only other compound in the staining solution. The CBB staning of the gel is done after SDS-PAGE and thorough washing of the gel in bidistilled water. By heating the gel during the washing and staining steps, the process can be finished faster and no toxic or hazardous compunds are evaporating. The staining of proteins occurs already within 1 minute after heating the gel in staining solution and is fully developed after 15-30 min with a slightly blue background that is destained completely by prolonged washing of the stained gel in bidistilled water, without affecting the stained protein bands.

A short protocol for protein staining with Coomassie Brilliant Blue (CBB) G-250 in polyacrylamide gels is described without using organic solvents or acetic acid as in the classical staining procedures with CBB.

无有机溶剂和醋酸，蛋白质与考马斯亮蓝G - 250凝胶染色

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents ...

Using the GELFREE 8100 Fractionation System for Molecular Weight-Based Fractionation with Liquid Phase Recovery

The GELFREE 8100 Fractionation System is a novel protein fractionation system designed to maximize protein recovery during molecular weight based fractionation. The system is comprised of single-use, 8-sample capacity cartridges and a benchtop GELFREE Fractionation Instrument. During separation, a constant voltage is applied between the anode and cathode reservoirs, and each protein mixture is electrophoretically driven from a loading chamber into a specially designed gel column gel. Proteins are concentrated into a tight band in a stacking gel, and separated based on their respective electrophoretic mobilities in a resolving gel. As proteins elute from the column, they are trapped and concentrated in liquid phase in the collection chamber, free of the gel. The instrument is then paused at specific time intervals, and fractions are collected using a pipette. This process is repeated until all desired fractions have been collected. If fewer than 8 samples are run on a cartridge, any unused chambers can be used in subsequent separations.
This novel technology facilitates the quick and simple separation of up to 8 complex protein mixtures simultaneously, and offers several advantages when compared to previously available fractionation methods. This system is capable of fractionating up to 1mg of total protein per channel, for a total of 8mg per cartridge. Intact proteins over a broad mass range are separated on the basis of molecular weight, retaining important physiochemical properties of the analyte. The liquid phase entrapment provides for high recovery while eliminating the need for band or spot cutting, making the fractionation process highly reproducible1.

The accompanying video describes the use of the GELFREE 8100 Fractionation System, which partitions complex protein samples on the basis of molecular weight and recovers the fractions in liquid phase. The video describes how the technology works, how it is used, and provides resultant data, with polyacrylamide gel electrophoresis analysis of fractionated bovine liver homogenate.

液相恢复GELFREE 8100分馏系统使用基于分子量分馏

The GELFREE 8100 Fractionation System is a novel protein fractionation system designed to maximize protein recovery during molecular weight based ...

In-vivo Detection of Protein-protein Interactions on Micro-patterned Surfaces

Unraveling the interaction network of molecules in-vivo is key to understanding the mechanisms that regulate cell function and metabolism. A multitude of methodological options for addressing molecular interactions in cells have been developed, but most of these methods suffer from being rather indirect and therefore hardly quantitative. On the contrary, a few high-end quantitative approaches were introduced, which however are difficult to extend to high throughput. To combine high throughput capabilities with the possibility to extract quantitative information, we recently developed a new concept for identifying protein-protein interactions (Schwarzenbacher et al., 2008). Here, we describe a detailed protocol for the design and the construction of this system which allows for analyzing interactions between a fluorophore-labeled protein ("prey") and a membrane protein ("bait") in-vivo. Cells are plated on micropatterned surfaces functionalized with antibodies against the bait exoplasmic domain. Bait-prey interactions are assayed via the redistribution of the fluorescent prey. The method is characterized by high sensitivity down to the level of single molecules, the capability to detect weak interactions, and high throughput capability, making it applicable as screening tool.

In-vivo Detection of Protein-protein Interactions on Micro-patterned Surfaces

This video shows experiments with subsequent analysis of protein-protein interactions by the use of micro-patterned surfaces. The approach offers the possibility to detect protein interactions in living cells and combines high throughput capabilities with the possibility to extract quantitative information.

微图案的表面上的蛋白质-蛋白质相互作用的体内检测

Unraveling the interaction network of molecules in-vivo is key to understanding the mechanisms that regulate cell function and metabolism. A multitude of ...

Detection of Protein Interactions in Plant using a Gateway Compatible Bimolecular Fluorescence Complementation (BiFC) System

We have developed a BiFC technique to test the interaction between two proteins in vivo. This is accomplished by splitting a yellow fluorescent protein (YFP) into two non-overlapping fragments. Each fragment is cloned in-frame to a gene of interest. These constructs can then be co-transformed into Nicotiana benthamiana via Agrobacterium mediated transformation, allowing the transit expression of fusion proteins. The reconstitution of YFP signal only occurs when the inquest proteins interact 1-7. To test and validate the protein-protein interactions, BiFC can be used together with yeast two hybrid (Y2H) assay. This may detect indirect interactions which can be overlooked in the Y2H. Gateway technology is a universal platform that enables researchers to shuttle the gene of interest (GOI) into as many expression and functional analysis systems as possible8,9. Both the orientation and reading frame can be maintained without using restriction enzymes or ligation to make expression-ready clones. As a result, one can eliminate all the re-sequencing steps to ensure consistent results throughout the experiments. We have created a series of Gateway compatible BiFC and Y2H vectors which provide researchers with easy-to-use tools to perform both BiFC and Y2H assays10. Here, we demonstrate the ease of using our BiFC system to test protein-protein interactions in N. benthamiana plants.

Detection of Protein Interactions in Plant using a Gateway Compatible Bimolecular Fluorescence Complementation BiFC System

We have developed a technique to test protein-protein interactions in plant. A yellow fluorescent protein (YFP) is split into two non-overlapping fragments. Each fragment is cloned in-frame to a gene of interest via Gateway system, enabling expression of fusion proteins. Reconstitution of YFP signal only occurs when the inquest proteins interact.

使用网关兼容双分子荧光互补（附设）系统在植物蛋白相互作用检测

We have developed a BiFC technique to test the interaction between two proteins in vivo. This is accomplished by splitting a yellow fluorescent protein ...

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

The production of recombinant membrane proteins for structural and functional studies remains technically challenging due to low levels of expression and the inherent instability of many membrane proteins once solubilized in detergents. A protocol is described that combines ligation independent cloning of membrane proteins as GFP fusions with expression in Escherichia coli detected by GFP fluorescence. This enables the construction and expression screening of multiple membrane protein/variants to identify candidates suitable for further investment of time and effort. The GFP reporter is used in a primary screen of expression by visualizing GFP fluorescence following SDS polyacrylamide gel electrophoresis (SDS-PAGE). Membrane proteins that show both a high expression level with minimum degradation as indicated by the absence of free GFP, are selected for a secondary screen. These constructs are scaled and a total membrane fraction prepared and solubilized in four different detergents. Following ultracentrifugation to remove detergent-insoluble material, lysates are analyzed by fluorescence detection size exclusion chromatography (FSEC). Monitoring the size exclusion profile by GFP fluorescence provides information about the mono-dispersity and integrity of the membrane proteins in different detergents. Protein: detergent combinations that elute with a symmetrical peak with little or no free GFP and minimum aggregation are candidates for subsequent purification. Using the above methodology, the heterologous expression in E. coli of SED (shape, elongation, division, and sporulation) proteins from 47 different species of bacteria was analyzed. These proteins typically have ten transmembrane domains and are essential for cell division. The results show that the production of the SEDs orthologues in E. coli was highly variable with respect to the expression levels and integrity of the GFP fusion proteins. The experiment identified a subset for further investigation.

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

A streamlined approach to screening for the expression of recombinant membrane proteins in Escherichia coli based on fusion to green fluorescent protein is presented.

膜蛋白的绿色荧光蛋白为基础的表达筛选<em&gt;大肠杆菌</em

The production of recombinant membrane proteins for structural and functional studies remains technically challenging due to low levels of expression and ...

Protein Purification Technique that Allows Detection of Sumoylation and Ubiquitination of Budding Yeast Kinetochore Proteins Ndc10 and Ndc80

Post-translational Modifications (PTMs), such as phosphorylation, methylation, acetylation, ubiquitination, and sumoylation, regulate the cellular function of many proteins. PTMs of kinetochore proteins that associate with centromeric DNA mediate faithful chromosome segregation to maintain genome stability. Biochemical approaches such as mass spectrometry and western blot analysis are most commonly used for identification of PTMs. Here, a protein purification method is described that allows the detection of both sumoylation and ubiquitination of the kinetochore proteins, Ndc10 and Ndc80, in Saccharomyces cerevisiae. A strain that expresses polyhistidine-Flag-tagged Smt3 (HF-Smt3) and Myc-tagged Ndc10 or Ndc80 was constructed and used for our studies. For detection of sumoylation, we devised a protocol to affinity purify His-tagged sumoylated proteins by using nickel beads and used western blot analysis with anti-Myc antibody to detect sumoylated Ndc10 and Ndc80. For detection of ubiquitination, we devised a protocol for immunoprecipitation of Myc-tagged proteins and used western blot analysis with anti-Ub antibody to show that Ndc10 and Ndc80 are ubiquitinated. Our results show that epitope tagged-protein of interest in the His-Flag tagged Smt3 strain facilitates the detection of multiple PTMs. Future studies should allow exploitation of this technique to identify and characterize protein interactions that are dependent on a specific PTM.

This manuscript describes the detection of sumoylation and ubiquitination of kinetochore proteins, Ndc10 and Ndc80, in the budding yeast Saccharomyces cerevisiae.

蛋白质纯化技术，可以检测SUMO化修饰和芽殖酵母蛋白动粒和Ndc10泛素Ndc80的

Post-translational Modifications (PTMs), such as phosphorylation, methylation, acetylation, ubiquitination, and sumoylation, regulate the cellular ...

Detection of Ligand-activated G Protein-coupled Receptor Internalization by Confocal Microscopy

Confocal laser scanning microscopy (CLSM) is an optical imaging technique for high-contrast imaging. It is a powerful approach to visualize fluorescent fusion proteins, such as green fluorescent protein (GFP), to determine their expression, localization, and function. The subcellular localization of target proteins is important for identification, characterization, and functional analyses. Internalization is one of the predominant mechanisms controlling G protein-coupled receptor (GPCR) signaling to ensure the appropriate cellular responses to stimuli. Here, we describe an experimental method to detect the subcellular localization and internalization of GPCR in HEK293 cells with confocal microscopy. In addition, this experiment provides some details about cell culture and transfection. This protocol is compatible with a variety of widely available fluorescent markers and is applicable to the visualization of the subcellular localization of a majority of proteins, as well as of the internalization of GPCR. This technique should enable researchers to efficiently manipulate GPCR gene expression in mammalian cell lines and should facilitate studies on GPCR subcellular localization and internalization.

This protocol describes confocal microscopy detection of G protein-coupled receptor (GPCR) internalization in mammalian cells. It includes the basic cell culture, transfection, and confocal microscopy procedure and provides an efficient and easily interpretable method to detect the subcellular localization and internalization of fusion-expressed GPCR.

配体激活的G蛋白偶联受体内在化的检测通过共聚焦显微镜

Confocal laser scanning microscopy (CLSM) is an optical imaging technique for high-contrast imaging. It is a powerful approach to visualize fluorescent ...

In Vivo Detection and Analysis of Rb Protein SUMOylation in Human Cells

The post-translational modifications of proteins are critical for the proper regulation of intracellular signal transduction. Among these modifications, small ubiquitin-related modifier (SUMO) is a ubiquitin-like protein that is covalently attached to the lysine residues of a variety of target proteins to regulate cellular processes, such as gene transcription, DNA repair, protein interaction and degradation, subcellular transport, and signal transduction. The most common approach to detecting protein SUMOylation is based on the expression and purification of recombinant tagged proteins in bacteria, allowing for an in vitro biochemical reaction which is simple and suitable for addressing mechanistic questions. However, due to the complexity of the process of SUMOylation in vivo, it is more challenging to detect and analyze protein SUMOylation in cells, especially when under endogenous conditions. A recent study by the authors of this paper revealed that endogenous retinoblastoma (Rb) protein, a tumor suppressor that is vital to the negative regulation of the cell cycle progression, is specifically SUMOylated at the early G1 phase. This paper describes a protocol for the detection and analysis of Rb SUMOylation under both endogenous and exogenous conditions in human cells. This protocol is appropriate for the phenotypical and functional investigation of the SUMO-modification of Rb, as well as many other SUMO-targeted proteins, in human cells.

In Vivo Detection and Analysis of Rb Protein SUMOylation in Human Cells

Small ubiquitin-related modifier (SUMO) family proteins are conjugated to the lysine residues of target proteins to regulate various cellular processes. This paper describes a protocol for the detection of retinoblastoma (Rb) protein SUMOylation under endogenous and exogenous conditions in human cells.

在体内人细胞 Rb 蛋白 sumo 的检测与分析

The post-translational modifications of proteins are critical for the proper regulation of intracellular signal transduction. Among these modifications, ...

Automated Detection and Analysis of Exocytosis

Timelapse TIRF microscopy of pH-sensitive GFP (pHluorin) attached to vesicle SNARE proteins is an effective method to visualize single vesicle exocytic events in cell culture. To perform an unbiased, efficient identification and analysis of such events, a computer-vision based approach was developed and implemented in MATLAB. The analysis pipeline consists of a cell segmentation and exocytic-event identification algorithm. The computer-vision approach includes tools for investigating multiple parameters of single events, including the half-life of fluorescence decay and peak &#916;F/F, as well as whole-cell analysis of the frequency of exocytosis. These and other parameters of fusion are used in a classification approach to distinguish distinct fusion modes. Here a newly built GUI performs the analysis pipeline from start to finish. Further adaptation of Ripley&#39;s K function in R Studio is used to distinguish between clustered, dispersed, or random occurrence of fusion events in both space and time.

We developed automated computer vision software to detect exocytic events marked by pH-sensitive fluorescent probes. Here, we demonstrate the use of a graphical user interface and RStudio to detect fusion events, analyze and display spatiotemporal parameters of fusion, and classify events into distinct fusion modes.

外分泌物的自动检测和分析

Timelapse TIRF microscopy of pH-sensitive GFP (pHluorin) attached to vesicle SNARE proteins is an effective method to visualize single vesicle exocytic ...