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Current Protocols in Protein Science最新文献

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pI Determination of Native Proteins In Biological Samples 生物样品中天然蛋白的测定
Current Protocols in Protein Science Pub Date : 2019-01-31 DOI: 10.1002/cpps.85
Shanmugasundaram Ganapathy-Kanniappan
{"title":"pI Determination of Native Proteins In Biological Samples","authors":"Shanmugasundaram Ganapathy-Kanniappan","doi":"10.1002/cpps.85","DOIUrl":"10.1002/cpps.85","url":null,"abstract":"<p>The electrophoretic mobility of a protein on an immobilized pH-gradient gel (IPG) depends upon its overall positive (acidic) or negative (basic) charge, the principle underlying the IEF technique. In isoelectrofocusing (IEF), a protein with a net positive or negative charge migrates through the pH gradient gel until it reaches the isoelectric point (pI), a pH at which it remains neutral. Thus, the pI of a protein indicates its net charge, a critical determinant of its stability/activity in a given milieu. Conventionally, the first-dimensional IPG-IEF is followed by a second dimension, by which the focused proteins are denatured/reduced and resolved on an SDS-PAGE gel for subsequent immunoblotting to verify the protein identity. The recent development of one-dimensional, vertical IEF followed by immunoblotting enabled concurrent analysis (pI determination) of multiple samples. The protocol described here outlines vertical IEF and immunoblotting under non-denaturing conditions to determine the pI of native proteins in biological samples. © 2019 by John Wiley &amp; Sons, Inc.</p>","PeriodicalId":10866,"journal":{"name":"Current Protocols in Protein Science","volume":"96 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2019-01-31","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpps.85","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"36916259","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 3
Issue Information TOC 发布信息TOC
Current Protocols in Protein Science Pub Date : 2019-01-22 DOI: 10.1002/cpps.79
{"title":"Issue Information TOC","authors":"","doi":"10.1002/cpps.79","DOIUrl":"10.1002/cpps.79","url":null,"abstract":"","PeriodicalId":10866,"journal":{"name":"Current Protocols in Protein Science","volume":"95 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2019-01-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpps.79","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"45154691","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
Expression and Preparation of a G-Protein-Coupled Cannabinoid Receptor CB2 for NMR Structural Studies g蛋白偶联大麻素受体CB2的表达和制备及其核磁共振结构研究
Current Protocols in Protein Science Pub Date : 2019-01-09 DOI: 10.1002/cpps.83
Alexei Yeliseev
{"title":"Expression and Preparation of a G-Protein-Coupled Cannabinoid Receptor CB2 for NMR Structural Studies","authors":"Alexei Yeliseev","doi":"10.1002/cpps.83","DOIUrl":"10.1002/cpps.83","url":null,"abstract":"<p>Cannabinoid receptor type II, or CB<sub>2</sub>, is an integral membrane protein that belongs to a large class of G-protein-coupled receptors (GPCR)s. CB<sub>2</sub> is a part of the endocannabinoid system, which plays an important role in the regulation of immune response, inflammation, and pain. Information about the structure and function of CB<sub>2</sub> is essential for the development of specific ligands targeting this receptor. We present here a methodology for recombinant expression of CB<sub>2</sub> and its stable isotope labeling, purification, and reconstitution into liposomes, in preparation for its characterization by nuclear magnetic resonance (NMR). Correctly folded, functional CB<sub>2</sub> labeled with [<sup>13</sup>C,<sup>15</sup>N]tryptophan or uniformly labeled with <sup>13</sup>C and <sup>15</sup>N is expressed in a medium of defined composition, under controlled aeration, pH, and temperature conditions. The receptor is purified by affinity chromatography and reconstituted into lipid bilayers in the form of proteoliposomes suitable for analysis by NMR spectroscopy. © 2019 by John Wiley &amp; Sons, Inc.</p>","PeriodicalId":10866,"journal":{"name":"Current Protocols in Protein Science","volume":"96 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2019-01-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpps.83","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"36890780","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 9
Issue Information TOC 发布信息TOC
Current Protocols in Protein Science Pub Date : 2018-10-29 DOI: 10.1002/cpps.78
{"title":"Issue Information TOC","authors":"","doi":"10.1002/cpps.78","DOIUrl":"10.1002/cpps.78","url":null,"abstract":"","PeriodicalId":10866,"journal":{"name":"Current Protocols in Protein Science","volume":"94 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2018-10-29","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpps.78","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"45243518","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
Proteome-Wide Analysis of Cysteine S-Sulfenylation Using a Benzothiazine-Based Probe 基于苯并噻嗪探针的半胱氨酸s -磺化蛋白组分析
Current Protocols in Protein Science Pub Date : 2018-10-12 DOI: 10.1002/cpps.76
Ling Fu, Keke Liu, Renan B. Ferreira, Kate S. Carroll, Jing Yang
{"title":"Proteome-Wide Analysis of Cysteine S-Sulfenylation Using a Benzothiazine-Based Probe","authors":"Ling Fu,&nbsp;Keke Liu,&nbsp;Renan B. Ferreira,&nbsp;Kate S. Carroll,&nbsp;Jing Yang","doi":"10.1002/cpps.76","DOIUrl":"10.1002/cpps.76","url":null,"abstract":"<p>Oxidation of a protein cysteinyl thiol (Cys-SH) to S-sulfenic acid (Cys-SOH) by a reactive oxygen species (e.g., hydrogen peroxide), which is termed protein S-sulfenylation, is a reversible post-translational modification that plays a crucial role in redox regulation of protein function in various biological processes. Due to its intrinsically labile nature, protein S-sulfenylation cannot be directly detected or analyzed. Chemoselective probing has been the method of choice for analyzing S-sulfenylated proteins either <i>in vitro</i> or <i>in situ</i>, as it allows stabilization and direct detection of this transient oxidative intermediate. However, it remains challenging to globally pinpoint the specific S-sulfenylated cysteine sites on complex proteomes and to quantify their dynamic changes upon oxidative stress. This unit describes how a benzothiazine-based chemoselective probe called BTD and mass spectrometry based chemoproteomics can be used to globally and site-specifically identify and quantify protein S-sulfenylation. © 2018 by John Wiley &amp; Sons, Inc.</p>","PeriodicalId":10866,"journal":{"name":"Current Protocols in Protein Science","volume":"95 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2018-10-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpps.76","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"36579109","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 41
Analysis of Cysteine Post Translational Modifications Using Organic Mercury Resin 有机汞树脂对半胱氨酸翻译后修饰的分析
Current Protocols in Protein Science Pub Date : 2018-10-03 DOI: 10.1002/cpps.69
Paschalis-Thomas Doulias, Neal S. Gould
{"title":"Analysis of Cysteine Post Translational Modifications Using Organic Mercury Resin","authors":"Paschalis-Thomas Doulias,&nbsp;Neal S. Gould","doi":"10.1002/cpps.69","DOIUrl":"10.1002/cpps.69","url":null,"abstract":"<p>The wide reactivity of the thiol group enables the formation of a variety of reversible, covalent modifications on cysteine residues. <i>S</i>-nitrosylation, like many other post-translational modifications, is site selective, reversible, and necessary for a wide variety of fundamental cellular processes. The overall abundance of <i>S</i>-nitrosylated proteins and reactivity of the nitrosyl group necessitates an enrichment strategy for accurate detection with adequate depth. Herein, a method is presented for the enrichment and detection of endogenous protein <i>S</i>-nitrosylation from complex mixtures of cell or tissue lysate utilizing organomercury resin. Minimal adaptations to the method also support the detection of either <i>S</i>-glutathionylation or <i>S</i>-acylation using the same enrichment platform. When coupled with high accuracy mass spectrometry, these methods enable a site-specific level of analysis, facilitating the curation comparable datasets of three separate cysteine post-translational modifications. © 2018 by John Wiley &amp; Sons, Inc.</p>","PeriodicalId":10866,"journal":{"name":"Current Protocols in Protein Science","volume":"94 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2018-10-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpps.69","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"36552885","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 6
Optimization of Protein Expression in Mammalian Cells 哺乳动物细胞中蛋白表达的优化
Current Protocols in Protein Science Pub Date : 2018-09-28 DOI: 10.1002/cpps.77
Molly Hunter, Ping Yuan, Divya Vavilala, Mark Fox
{"title":"Optimization of Protein Expression in Mammalian Cells","authors":"Molly Hunter,&nbsp;Ping Yuan,&nbsp;Divya Vavilala,&nbsp;Mark Fox","doi":"10.1002/cpps.77","DOIUrl":"10.1002/cpps.77","url":null,"abstract":"<p>Recombinant proteins, such as monoclonal antibodies, are produced in mammalian cell lines to introduce proper protein folding and post-translational modifications, which are essential for full biological activity. In both the industrial and academic environments, the use of recombinant proteins varies widely and, with it, the method of production. The amount of an antibody needed for a toxicity study is far greater than that needed by a research lab performing cellular assays, and the amount of effort put into the development of the protein will vary accordingly. There is no universal strategy for mammalian expression systems, and scientists often struggle to develop a suitable process from the myriad of choices at each step. Here, we elaborate on the various obstacles encountered when planning high-yield experiments to produce the recombinant proteins of interest. © 2018 by John Wiley &amp; Sons, Inc.</p>","PeriodicalId":10866,"journal":{"name":"Current Protocols in Protein Science","volume":"95 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2018-09-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpps.77","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"36533165","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 91
Quantitative Comparison of Proteomes Using SILAC 使用SILAC定量比较蛋白质组
Current Protocols in Protein Science Pub Date : 2018-09-20 DOI: 10.1002/cpps.74
Jingjing Deng, Hediye Erdjument-Bromage, Thomas A. Neubert
{"title":"Quantitative Comparison of Proteomes Using SILAC","authors":"Jingjing Deng,&nbsp;Hediye Erdjument-Bromage,&nbsp;Thomas A. Neubert","doi":"10.1002/cpps.74","DOIUrl":"10.1002/cpps.74","url":null,"abstract":"<p>Stable isotope labeling by amino acids in cell culture (SILAC) has become very popular as a quantitative proteomic method since it was firstly introduced by Matthias Mann's group in 2002. It is a metabolic labeling strategy in which isotope-labeled amino acids are metabolically incorporated in vivo into proteins during translation. After natural (light) or heavy amino acid incorporation, differentially labeled samples are mixed immediately after cell lysis and before any further processing, which minimizes quantitative errors caused by handling different samples in parallel. In this unit, we describe protocols for basic duplex SILAC, triplex SILAC for use in nondividing cells such as neurons, and for measuring amounts of newly synthesized proteins. © 2018 by John Wiley &amp; Sons, Inc.</p>","PeriodicalId":10866,"journal":{"name":"Current Protocols in Protein Science","volume":"95 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2018-09-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpps.74","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"36508056","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 25
Characterization of Membrane Proteins Using Cryo-Electron Microscopy 膜蛋白的低温电镜表征
Current Protocols in Protein Science Pub Date : 2018-09-10 DOI: 10.1002/cpps.72
Vanessa Carvalho, Joachim W. Pronk, Andreas H. Engel
{"title":"Characterization of Membrane Proteins Using Cryo-Electron Microscopy","authors":"Vanessa Carvalho,&nbsp;Joachim W. Pronk,&nbsp;Andreas H. Engel","doi":"10.1002/cpps.72","DOIUrl":"10.1002/cpps.72","url":null,"abstract":"<p>The steep increase of atomic scale structures determined by 3D cryo-electron microscopy (EM) deposited in the EMDataBank documents progress of a methodology that was frustratingly slow ten years ago. While sample vitrification on grids has been successfully used in all EM laboratories for decades, beam damage remains a road block. Developments in instrumentation and software to exploit the information carried by elastically scattered electrons made the task to achieve atomic scale resolution easier. This together with the development of fast single electron detecting cameras has resulted in unprecedented possibilities for structure determination by 3D cryo-EM. With such technologies in place, the purification of membrane protein complexes in a functional state is key to collecting atomic scale structural information and insight into the chemistry of physiological processes. Therefore, we focus here on the preparation of membrane proteins for structural analyses by 3D cryo-EM and the data acquisition of such vitrified samples. © 2018 by John Wiley &amp; Sons, Inc.</p>","PeriodicalId":10866,"journal":{"name":"Current Protocols in Protein Science","volume":"94 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2018-09-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpps.72","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"36476862","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 1
Yeast Two-Hybrid Assay to Identify Interacting Proteins 酵母双杂交法鉴定相互作用蛋白
Current Protocols in Protein Science Pub Date : 2018-08-21 DOI: 10.1002/cpps.70
Aurora Paiano, Azzurra Margiotta, Maria De Luca, Cecilia Bucci
{"title":"Yeast Two-Hybrid Assay to Identify Interacting Proteins","authors":"Aurora Paiano,&nbsp;Azzurra Margiotta,&nbsp;Maria De Luca,&nbsp;Cecilia Bucci","doi":"10.1002/cpps.70","DOIUrl":"10.1002/cpps.70","url":null,"abstract":"<p>This article describes the general method to perform the classical two-hybrid system. Although it has already been more than 25 years since this technique was developed, it still represents one of the best and most inexpensive, time saving, and straightforward methods to identify and study protein-protein interactions. Indeed, this system can be easily used to identify interacting proteins for a given protein, to check interactions between two known proteins, or to map interacting domains. Most of the interactions revealed using the two-hybrid assay have been proven to be binary direct interactions. Data comparison with other systems, such as mass spectrometry, have demonstrated that this system is at least as reliable. In fact, its use is increasing with time, and at present numerous variants of the yeast two-hybrid assay have been developed, including high-throughput systems that promote the generation of a proteome-scale map of protein-protein interactions in specific system. © 2018 by John Wiley &amp; Sons, Inc.</p>","PeriodicalId":10866,"journal":{"name":"Current Protocols in Protein Science","volume":"95 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2018-08-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpps.70","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"36416978","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 34
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