发布求助
文献互助 智能选刊 最新文献

Current Protocols in Molecular Biology最新文献

筛选
英文 中文
Growth of E. coli in Liquid Medium 大肠杆菌在液体培养基中的生长
Current Protocols in Molecular Biology Pub Date : 2018-11-09 DOI: 10.1002/cpmb.81
Karen L. Elbing, Roger Brent
{"title":"Growth of E. coli in Liquid Medium","authors":"Karen L. Elbing,&nbsp;Roger Brent","doi":"10.1002/cpmb.81","DOIUrl":"10.1002/cpmb.81","url":null,"abstract":"<p>We describe the procedure for inoculating overnight (starter) cultures of <i>E. coli</i> from a single colony, along with considerations for growing larger cultures. We also include two methods for monitoring the number of cells per unit volume (density) of liquid cultures using a spectrophotometer and a hemacytometer or “count slide.” © 2018 by John Wiley &amp; Sons, Inc.</p>","PeriodicalId":10734,"journal":{"name":"Current Protocols in Molecular Biology","volume":"125 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2018-11-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpmb.81","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"36710481","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: 7
Two-Step Co-Immunoprecipitation (TIP) 两步共免疫沉淀(TIP)
Current Protocols in Molecular Biology Pub Date : 2018-10-30 DOI: 10.1002/cpmb.80
Maria Rita Sciuto, Valeria Coppola, Gioacchin Iannolo, Ruggero De Maria, Tobias L. Haas
{"title":"Two-Step Co-Immunoprecipitation (TIP)","authors":"Maria Rita Sciuto,&nbsp;Valeria Coppola,&nbsp;Gioacchin Iannolo,&nbsp;Ruggero De Maria,&nbsp;Tobias L. Haas","doi":"10.1002/cpmb.80","DOIUrl":"10.1002/cpmb.80","url":null,"abstract":"<p>In the past few decades, numerous approaches have been developed to investigate protein-protein and protein-nucleic acid interactions (PPIs and PNIs). Affinity purification methods such as co-immunoprecipitation (co-IP) are commonly used to detect and isolate the macromolecular complexesresulting from these interactions. In this article, we describe a two-step co-immunoprecipitation (TIP) technique. As compared to standard co-IP, TIP provides increased specificity in the isolation of PPIs or PNIs under native expression conditions, dramatically reducing the abundance of nonspecific binders and thus facilitating downstream analyses of the interaction complexes. Here, we report a detailed TIP procedure that we used to purify a protein-protein complex from Burkitt lymphoma cells and from primary human CD4<sup>+</sup> T cells. In addition, this unit describes an application of TIP for the isolation of transcription-factor-bound chromatin. © 2018 by John Wiley &amp; Sons, Inc.</p>","PeriodicalId":10734,"journal":{"name":"Current Protocols in Molecular Biology","volume":"125 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2018-10-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpmb.80","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"36629651","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: 2
Refolding Proteins from Inclusion Bodies using Differential Scanning Fluorimetry Guided (DGR) Protein Refolding and MeltTraceur Web 利用差示扫描荧光法引导(DGR)蛋白质重折叠和熔融示踪网从包涵体中重折叠蛋白质
Current Protocols in Molecular Biology Pub Date : 2018-10-29 DOI: 10.1002/cpmb.78
Mark E. Lee, Xiaoyi Dou, Yingmin Zhu, Kevin J. Phillips
{"title":"Refolding Proteins from Inclusion Bodies using Differential Scanning Fluorimetry Guided (DGR) Protein Refolding and MeltTraceur Web","authors":"Mark E. Lee,&nbsp;Xiaoyi Dou,&nbsp;Yingmin Zhu,&nbsp;Kevin J. Phillips","doi":"10.1002/cpmb.78","DOIUrl":"10.1002/cpmb.78","url":null,"abstract":"<p>Differential Scanning Fluorimetry Guided Refolding (DGR) is a simple methodology that can be used to rapidly screen for and identify conditions capable of accurately refolding protein preparations, such as those obtained from <i>Escherichia coli</i> inclusion bodies. It allows for the production in <i>E. coli</i> of functional proteins that would otherwise require far more expensive production methods. This unit describes how to set up a DGR refolding assay, perform DGR refolding trials in microplate format, use MeltTraceur Web software to interactively analyze the resulting data, scale-up protein production via refolding, and lastly, validate that the protein is properly folded. © 2018 by John Wiley &amp; Sons, Inc.</p>","PeriodicalId":10734,"journal":{"name":"Current Protocols in Molecular Biology","volume":"125 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2018-10-29","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpmb.78","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"36626477","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
Polysome Profiling Analysis of mRNA and Associated Proteins Engaged in Translation 参与翻译的mRNA和相关蛋白的多聚体分析
Current Protocols in Molecular Biology Pub Date : 2018-10-29 DOI: 10.1002/cpmb.79
Eric S. Pringle, Craig McCormick, Zhenyu Cheng
{"title":"Polysome Profiling Analysis of mRNA and Associated Proteins Engaged in Translation","authors":"Eric S. Pringle,&nbsp;Craig McCormick,&nbsp;Zhenyu Cheng","doi":"10.1002/cpmb.79","DOIUrl":"10.1002/cpmb.79","url":null,"abstract":"<p>Post-transcriptional regulation is an important aspect of the control of gene expression. mRNAs are translated with variable efficiencies, and these efficiencies can change rapidly during adaptation to diverse environmental factors, including cellular stresses and microbial infections. Polysome profiling analysis utilizes ultracentrifugation to isolate complexes of mRNAs in the process of translation and corresponding proteins on the basis of density. Here we describe polysome profiling analysis using a continuous ultraviolet spectrophotometer and a gradient fractionator. We provide protocols for processing sucrose gradient fractions for isolation of RNA for RT-qPCR analysis and isolation of protein for SDS-PAGE and immunoblot analysis. © 2018 by John Wiley &amp; Sons, Inc.</p>","PeriodicalId":10734,"journal":{"name":"Current Protocols in Molecular Biology","volume":"125 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2018-10-29","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpmb.79","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"36626476","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: 47
Targeted Profiling of RNA Translation RNA翻译的靶向分析
Current Protocols in Molecular Biology Pub Date : 2018-10-22 DOI: 10.1002/cpmb.71
Ben B. Li, Changli Qian, Thomas M. Roberts, Jean J. Zhao
{"title":"Targeted Profiling of RNA Translation","authors":"Ben B. Li,&nbsp;Changli Qian,&nbsp;Thomas M. Roberts,&nbsp;Jean J. Zhao","doi":"10.1002/cpmb.71","DOIUrl":"10.1002/cpmb.71","url":null,"abstract":"<p>This unit describes a reverse transcription-quantitative PCR (RT-qPCR)–based method for gene-targeted measurement of RNA translation levels. The method includes washing and lysing cells with a buffer containing cycloheximide to enrich ribosomal accumulation at translation initiation sites (TIS), followed by enzymatic treatment to generate ribosomal footprints, reverse transcription targeted towards TIS of specific transcripts of interest to generate complementary DNA (cDNA), and qPCR to measure the abundance of these footprints. This method enables time- and cost-effective assessment of changes in translation levels across focused panels of genes and across numerous samples. © 2018 by John Wiley &amp; Sons, Inc.</p>","PeriodicalId":10734,"journal":{"name":"Current Protocols in Molecular Biology","volume":"125 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2018-10-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpmb.71","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"36595031","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 问题信息TOC
Current Protocols in Molecular Biology Pub Date : 2018-10-01 DOI: 10.1002/cpmb.73
{"title":"Issue Information TOC","authors":"","doi":"10.1002/cpmb.73","DOIUrl":"https://doi.org/10.1002/cpmb.73","url":null,"abstract":"","PeriodicalId":10734,"journal":{"name":"Current Protocols in Molecular Biology","volume":"124 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2018-10-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpmb.73","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"72137094","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
Whole Genome Next-Generation Sequencing Mutation Identification in Pseudomonas aeruginosa 铜绿假单胞菌全基因组下一代测序突变鉴定
Current Protocols in Molecular Biology Pub Date : 2018-09-21 DOI: 10.1002/cpmb.69
Murat Cetinbas, Shen Yu, Ruslan I. Sadreyev
{"title":"Whole Genome Next-Generation Sequencing Mutation Identification in Pseudomonas aeruginosa","authors":"Murat Cetinbas,&nbsp;Shen Yu,&nbsp;Ruslan I. Sadreyev","doi":"10.1002/cpmb.69","DOIUrl":"10.1002/cpmb.69","url":null,"abstract":"<p>Identification of spontaneous or chemically induced bacterial mutations is a powerful tool for investigation of molecular mechanisms, including the mechanism of action of novel antibiotics. However, a major bottleneck to this approach has been the identification of the causative mutation underlying a phenotype of interest. Until recently, this has required time-consuming genetic analysis. However, the advent of relatively inexpensive and rapid next-generation sequencing (NGS) technologies has revolutionized the correlation of bacterial phenotypes and genotypes. In this article we describe a simple bioinformatics pipeline to identify differences between sequenced bacterial genomes. We also describe the procedures involved in growing, extracting, and purifying DNA, and preparation of sequencing libraries for one bacterial species, <i>Pseudomonas aeruginosa</i>. Similar protocols will be applicable to other bacterial species. © 2018 by John Wiley &amp; Sons, Inc.</p>","PeriodicalId":10734,"journal":{"name":"Current Protocols in Molecular Biology","volume":"124 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2018-09-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpmb.69","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"36508100","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
RNA-seq: Basic Bioinformatics Analysis RNA-seq:基础生物信息学分析
Current Protocols in Molecular Biology Pub Date : 2018-09-17 DOI: 10.1002/cpmb.68
Fei Ji, Ruslan I. Sadreyev
{"title":"RNA-seq: Basic Bioinformatics Analysis","authors":"Fei Ji,&nbsp;Ruslan I. Sadreyev","doi":"10.1002/cpmb.68","DOIUrl":"10.1002/cpmb.68","url":null,"abstract":"<p>Quantitative analysis of gene expression is crucial for understanding the molecular mechanisms underlying genome regulation. RNA-seq is a powerful platform for comprehensive investigation of the transcriptome. In this unit, we present a general bioinformatics workflow for the quantitative analysis of RNA-seq data and describe a few current publicly available computational tools applicable at various steps of this workflow. These tools comprise a pipeline for quality assessment and quantitation of RNA-seq data that starts from raw sequencing files and is focused on the identification and analysis of genes that are differentially expressed between biological conditions. © 2018 by John Wiley &amp; Sons, Inc.</p>","PeriodicalId":10734,"journal":{"name":"Current Protocols in Molecular Biology","volume":"124 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2018-09-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpmb.68","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"36495045","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: 36
RNAi Screening: Automated High-Throughput Liquid RNAi Screening in Caenorhabditis elegans RNAi筛选:秀丽隐杆线虫的自动高通量液体RNAi筛选
Current Protocols in Molecular Biology Pub Date : 2018-09-11 DOI: 10.1002/cpmb.65
Sakthimala Jagadeesan, Abdul Hakkim
{"title":"RNAi Screening: Automated High-Throughput Liquid RNAi Screening in Caenorhabditis elegans","authors":"Sakthimala Jagadeesan,&nbsp;Abdul Hakkim","doi":"10.1002/cpmb.65","DOIUrl":"10.1002/cpmb.65","url":null,"abstract":"<p>RNAi is a powerful reverse genetics tool that has revolutionized genetic studies in model organisms. The bacteriovorous nematode <i>Caenorhabditis elegans</i> can be genetically manipulated by feeding it an <i>Escherichia coli</i> strain that expresses a double-stranded RNA (dsRNA) corresponding to a <i>C. elegans</i> gene, which leads to systemic silencing of the gene. This unit describes protocols for performing an automated high-throughput RNAi screen utilizing a full-genome <i>C. elegans</i> RNAi library. The protocols employ liquid-handling robotics and 96-well plates. © 2018 by John Wiley &amp; Sons, Inc.</p>","PeriodicalId":10734,"journal":{"name":"Current Protocols in Molecular Biology","volume":"124 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2018-09-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpmb.65","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"36479708","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: 5
Plate Design for and Cherry Picking of Bacterial RNAi Clones for Systematic Error Detection in High-Throughput Caenorhabditis elegans RNAi Screens 高通量秀丽隐杆线虫RNAi筛选系统错误检测细菌RNAi克隆的平板设计与筛选
Current Protocols in Molecular Biology Pub Date : 2018-09-07 DOI: 10.1002/cpmb.70
Sakthimala Jagadeesan, Abdul Hakkim
{"title":"Plate Design for and Cherry Picking of Bacterial RNAi Clones for Systematic Error Detection in High-Throughput Caenorhabditis elegans RNAi Screens","authors":"Sakthimala Jagadeesan,&nbsp;Abdul Hakkim","doi":"10.1002/cpmb.70","DOIUrl":"10.1002/cpmb.70","url":null,"abstract":"<p>Automated or semi-automated high-throughput RNAi screens are highly prone to systematic errors because of multistep repetitive protocols and extensive use of automated instruments. A well-designed RNAi library can help detect and minimize systematic human and robotic errors. In this unit, we describe how to design an RNAi bacterial library for use in conjunction with the well-studied nematode <i>Caenorhabditis elegans</i> for automated phenotypic screens. We provide strategies to design and assemble RNAi libraries to reduce or eliminate systematic errors. These strategies serve as a good quality-control check and facilitate obtaining high-quality data from a genome-wide and sub-library RNAi screen. © 2018 by John Wiley &amp; Sons, Inc.</p>","PeriodicalId":10734,"journal":{"name":"Current Protocols in Molecular Biology","volume":"124 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2018-09-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpmb.70","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"36472087","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: 0
首页 上一页 下一页 尾页
0
×
引用
GB/T 7714-2015
复制
MLA
复制
APA
复制
导出至
BibTeX EndNote RefMan NoteFirst NoteExpress
×
提示
您的信息不完整，为了账户安全，请先补充。
现在去补充
×
提示
您因"违规操作"
具体请查看互助需知
我知道了
×
提示
现在去查看 取消
×
提示
确定
请完成安全验证×
相关产品
×
本文献相关产品
联系我们：info@booksci.cn Book学术提供免费学术资源搜索服务，方便国内外学者检索中英文文献。致力于提供最便捷和优质的服务体验。 Copyright © 2023 布克学术 All rights reserved.
京ICP备2023020795号-1
ghs 京公网安备 11010802042870号
Book学术文献互助
Book学术文献互助群
群 号：481959085
Book学术官方微信