Methods In Molecular Biology Directed Evolution Library Creation Methods And Protocols

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Directed Evolution Library Creation

Author : Elizabeth M. J. Gillam
Publisher :
ISBN 13 : 9781493910533
Total Pages : 369 pages
Book Rating : 4.9/5 (15 download)

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Book Synopsis Directed Evolution Library Creation by : Elizabeth M. J. Gillam

Download or read book Directed Evolution Library Creation written by Elizabeth M. J. Gillam and published by . This book was released on 2014 with total page 369 pages. Available in PDF, EPUB and Kindle. Book excerpt: Directed Evolution Library Creation: Methods and Protocols, Second Edition presents user-friendly protocols for both proven strategies and cutting-edge approaches for the creation of mutant gene libraries for directed evolution. As well as experimental methods, information on current computational approaches is provided in a user-friendly format that will allow researchers to make informed choices without needing to comprehend the full technical details of each algorithm. Directed evolution has become a fundamental approach for engineering proteins to enhance activity and explore structure-function relationships, and has supported the rapid development of the field of synthetic biology over the last decade. Divided into three convenient sections, topics include point mutagenesis strategies, recombinatorial methods wherein genetic diversity is sourced from multiple parental genes that are combined via either homology-dependent or -independent techniques, and a variety of computational methods to guide the design and analysis of mutant libraries. Written in the successful Methods in Molecular Biology series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible protocols, and notes on troubleshooting and avoiding known pitfalls. Authoritative and easily accessible, Directed Evolution Library Creation: Methods and Protocols, Second Edition will serve as a reliable manual for both novice and experienced protein engineers and synthetic biologists and will enable further technical innovation and the exploitation of directed evolution for a deeper understanding of protein design and function.

Methods in Molecular Biology: Directed evolution library creation: methods and protocols

Author : John M. Walker
Publisher :
ISBN 13 :
Total Pages : pages
Book Rating : 4.:/5 (84 download)

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Book Synopsis Methods in Molecular Biology: Directed evolution library creation: methods and protocols by : John M. Walker

Download or read book Methods in Molecular Biology: Directed evolution library creation: methods and protocols written by John M. Walker and published by . This book was released on 1984 with total page pages. Available in PDF, EPUB and Kindle. Book excerpt:

Directed Evolution

Author : Andrew Currin
Publisher : Springer Nature
ISBN 13 : 1071621521
Total Pages : 281 pages
Book Rating : 4.0/5 (716 download)

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Book Synopsis Directed Evolution by : Andrew Currin

Download or read book Directed Evolution written by Andrew Currin and published by Springer Nature. This book was released on 2022-06-21 with total page 281 pages. Available in PDF, EPUB and Kindle. Book excerpt: This volume explores the latest techniques used by researchers to study directed evolution (DE) at each stage of the Design-Build-Test-Learn cycle. Chapters in this book cover topics such as designing overlap extension PCR primers for protein mutagenesis; antha-guided automation of Darwin assembly for the construction of bespoke gene libraries; rapid cloning of random mutagenesis libraries using PTO-Quickstep; and DE of glycosyltransferases by a single-cell screening method. Written in the highly successful Methods in Molecular Biology series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls. Cutting-edge and comprehensive, Directed Evolution: Methods and Protocols is a valuable resource for scientists and researchers who are interested in learning more about this field and incorporating these studies into new experimental workflows.

Directed Evolution Library Creation

Author : Frances H. Arnold
Publisher : Humana Press
ISBN 13 : 9781617374715
Total Pages : 0 pages
Book Rating : 4.3/5 (747 download)

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Book Synopsis Directed Evolution Library Creation by : Frances H. Arnold

Download or read book Directed Evolution Library Creation written by Frances H. Arnold and published by Humana Press. This book was released on 2010-11-10 with total page 0 pages. Available in PDF, EPUB and Kindle. Book excerpt: Biological systems are very special substrates for engineering—uniquely the products of evolution, they are easily redesigned by similar approaches. A simple algorithm of iterative cycles of diversification and selection, evolution works at all scales, from single molecules to whole ecosystems. In the little more than a decade since the first reported applications of evolutionary design to enzyme engineering, directed evolution has matured to the point where it now represents the centerpiece of industrial biocatalyst development and is being practiced by thousands of academic and industrial scientists in com- nies and universities around the world. The appeal of directed evolution is easy to understand: it is conceptually straightforward, it can be practiced without any special instrumentation and, most important, it frequently yields useful solutions, many of which are totally unanticipated. Directed evolution has r- dered protein engineering readily accessible to a broad audience of scientists and engineers who wish to tailor a myriad of protein properties, including th- mal and solvent stability, enzyme selectivity, specific activity, protease s- ceptibility, allosteric control of protein function, ligand binding, transcriptional activation, and solubility. Furthermore, the range of applications has expanded to the engineering of more complex functions such as those performed by m- tiple proteins acting in concert (in biosynthetic pathways) or as part of mac- molecular complexes and biological networks.

Directed Enzyme Evolution

Author : Frances H. Arnold
Publisher : Springer Science & Business Media
ISBN 13 : 1592593968
Total Pages : 381 pages
Book Rating : 4.5/5 (925 download)

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Book Synopsis Directed Enzyme Evolution by : Frances H. Arnold

Download or read book Directed Enzyme Evolution written by Frances H. Arnold and published by Springer Science & Business Media. This book was released on 2008-02-02 with total page 381 pages. Available in PDF, EPUB and Kindle. Book excerpt: Directed evolution comprises two distinct steps that are typically applied in an iterative fashion: (1) generating molecular diversity and (2) finding among the ensemble of mutant sequences those proteins that perform the desired fu- tion according to the specified criteria. In many ways, the second step is the most challenging. No matter how cleverly designed or diverse the starting library, without an effective screening strategy the ability to isolate useful clones is severely diminished. The best screens are (1) high throughput, to increase the likelihood that useful clones will be found; (2) sufficiently sen- tive (i. e. , good signal to noise) to allow the isolation of lower activity clones early in evolution; (3) sufficiently reproducible to allow one to find small improvements; (4) robust, which means that the signal afforded by active clones is not dependent on difficult-to-control environmental variables; and, most importantly, (5) sensitive to the desired function. Regarding this last point, almost anyone who has attempted a directed evolution experiment has learned firsthand the truth of the dictum “you get what you screen for. ” The protocols in Directed Enzyme Evolution describe a series of detailed p- cedures of proven utility for directed evolution purposes. The volume begins with several selection strategies for enzyme evolution and continues with assay methods that can be used to screen enzyme libraries. Genetic selections offer the advantage that functional proteins can be isolated from very large libraries s- ply by growing a population of cells under selective conditions.

Directed Enzyme Evolution

Author : Frances H. Arnold
Publisher : Humana Press
ISBN 13 : 9781588292865
Total Pages : 383 pages
Book Rating : 4.2/5 (928 download)

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Book Synopsis Directed Enzyme Evolution by : Frances H. Arnold

Download or read book Directed Enzyme Evolution written by Frances H. Arnold and published by Humana Press. This book was released on 2003-05-16 with total page 383 pages. Available in PDF, EPUB and Kindle. Book excerpt: Directed evolution comprises two distinct steps that are typically applied in an iterative fashion: (1) generating molecular diversity and (2) finding among the ensemble of mutant sequences those proteins that perform the desired fu- tion according to the specified criteria. In many ways, the second step is the most challenging. No matter how cleverly designed or diverse the starting library, without an effective screening strategy the ability to isolate useful clones is severely diminished. The best screens are (1) high throughput, to increase the likelihood that useful clones will be found; (2) sufficiently sen- tive (i. e. , good signal to noise) to allow the isolation of lower activity clones early in evolution; (3) sufficiently reproducible to allow one to find small improvements; (4) robust, which means that the signal afforded by active clones is not dependent on difficult-to-control environmental variables; and, most importantly, (5) sensitive to the desired function. Regarding this last point, almost anyone who has attempted a directed evolution experiment has learned firsthand the truth of the dictum “you get what you screen for. ” The protocols in Directed Enzyme Evolution describe a series of detailed p- cedures of proven utility for directed evolution purposes. The volume begins with several selection strategies for enzyme evolution and continues with assay methods that can be used to screen enzyme libraries. Genetic selections offer the advantage that functional proteins can be isolated from very large libraries s- ply by growing a population of cells under selective conditions.

Protein Engineering Protocols

Author : Kristian Müller
Publisher : Springer Science & Business Media
ISBN 13 : 1597451878
Total Pages : 318 pages
Book Rating : 4.5/5 (974 download)

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Book Synopsis Protein Engineering Protocols by : Kristian Müller

Download or read book Protein Engineering Protocols written by Kristian Müller and published by Springer Science & Business Media. This book was released on 2007-10-26 with total page 318 pages. Available in PDF, EPUB and Kindle. Book excerpt: Protein engineering is a fascinating mixture of molecular biology, protein structure analysis, computation, and biochemistry, with the goal of developing useful or valuable proteins. Protein Engineering Protocols will consider the two general, but not mutually exclusive, strategies for protein engineering. The first is known as rational design, in which the scientist uses detailed knowledge of the structure and function of the protein to make desired changes. The s- ond strategy is known as directed evolution. In this case, random mutagenesis is applied to a protein, and selection or screening is used to pick out variants that have the desired qualities. By several rounds of mutation and selection, this method mimics natural evolution. An additional technique known as DNA shuffling mixes and matches pieces of successful variants to produce better results. This process mimics recombination that occurs naturally during sexual reproduction. The first section of Protein Engineering Protocols describes rational p- tein design strategies, including computational methods, the use of non-natural amino acids to expand the biological alphabet, as well as impressive examples for the generation of proteins with novel characteristics. Although procedures for the introduction of mutations have become routine, predicting and und- standing the effects of these mutations can be very challenging and requires profound knowledge of the system as well as protein structures in general.

Evolutionary Methods in Biotechnology

Author : Susanne Brakmann
Publisher : John Wiley & Sons
ISBN 13 : 3527604626
Total Pages : 227 pages
Book Rating : 4.5/5 (276 download)

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Book Synopsis Evolutionary Methods in Biotechnology by : Susanne Brakmann

Download or read book Evolutionary Methods in Biotechnology written by Susanne Brakmann and published by John Wiley & Sons. This book was released on 2006-03-06 with total page 227 pages. Available in PDF, EPUB and Kindle. Book excerpt: Miniturization and high throughput assay technology have brought the power of molecular evolution to the bioscience laboratory. Applied wisely, the evolutionary approach can quickly yield the desired result even where other methods have failed. From library generation by random or directed mutagenesis to screening and selection techniques -- the crucial steps for successful evolutionary biotechnology are described in detail in this practical guide that also includes valuable troubleshooting hints on frequently encountered problems. Modern methods for the surface display of peptides and proteins, selective enrichment of nucleic acid aptamers and high-throughput screening of industrial biocatalysts are explained, and computer-based methods for in silico protein and RNA engineering are described as an alternative to in vitro approaches. A special section covers the patenting regulations with regard to biotechnological innovations derived from directed evolution. As an added bonus, a CD-ROM is included that contains software tools for library design, selection of mutagenesis positions, and various predictive algorithms. In short, this practice oriented handbook is an indispensable tool for every scientist working in this interdisciplinary research area.

PCR Protocols

Author : John M. S. Bartlett
Publisher : Springer Science & Business Media
ISBN 13 : 1592593844
Total Pages : 1083 pages
Book Rating : 4.5/5 (925 download)

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Book Synopsis PCR Protocols by : John M. S. Bartlett

Download or read book PCR Protocols written by John M. S. Bartlett and published by Springer Science & Business Media. This book was released on 2008-02-03 with total page 1083 pages. Available in PDF, EPUB and Kindle. Book excerpt: In this new edition, the editors have thoroughly updated and dramatically expanded the number of protocols to take advantage of the newest technologies used in all branches of research and clinical medicine today. These proven methods include real time PCR, SNP analysis, nested PCR, direct PCR, and long range PCR. Among the highlights are chapters on genome profiling by SAGE, differential display and chip technologies, the amplification of whole genome DNA by random degenerate oligonucleotide PCR, and the refinement of PCR methods for the analysis of fragmented DNA from fixed tissues. Each fully tested protocol is described in step-by-step detail by an established expert in the field and includes a background introduction outlining the principle behind the technique, equipment and reagent lists, tips on trouble shooting and avoiding known pitfalls, and, where needed, a discussion of the interpretation and use of results.

Generation of cDNA Libraries

Author : Shao-Yao Ying
Publisher : Springer Science & Business Media
ISBN 13 : 1592593593
Total Pages : 329 pages
Book Rating : 4.5/5 (925 download)

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Book Synopsis Generation of cDNA Libraries by : Shao-Yao Ying

Download or read book Generation of cDNA Libraries written by Shao-Yao Ying and published by Springer Science & Business Media. This book was released on 2008-02-03 with total page 329 pages. Available in PDF, EPUB and Kindle. Book excerpt: Since its invention and subsequent development nearly 20 years ago, po- merase chain reaction (PCR) has been extensively utilized to identify numerous gene probes in vitro and in vivo. However, attempts to generate complete and full-length complementary cDNA libraries were, for the most part, fruitless and remained elusive until the last decade, when simple and rapid methods were developed. With current decoding and potential application of human genome information to genechips, there are urgent needs for identification of functional significance of these decoded gene sequences. Inherent in bringing these app- cations to fruition is the need to generate a complete and full-length cDNA library for potential functional assays of specific gene sequences. Generation of cDNA Libraries: Methods and Protocols serves as a laboratory manual on the evolution of generation of cDNA libraries, covering both ba- ground information and step-by-step practical laboratory recipes for which p- tocols, reagents, operational tips, instrumentation, and other requirements are detailed. The first chapter of the book is an overview of the basics of generating cDNA libraries, which include the following: (a) the definition of a cDNA library, (b) different kinds of cDNA libraries, (c) differences between methods for cDNA library generation using conventional approaches and novel stra- gies, including reverse generation of RNA repertoires from cDNA libraries, and (d) the quality of cDNA libraries.

E. coli Plasmid Vectors

Author : Nicola Casali
Publisher : Springer Science & Business Media
ISBN 13 : 1592594093
Total Pages : 311 pages
Book Rating : 4.5/5 (925 download)

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Book Synopsis E. coli Plasmid Vectors by : Nicola Casali

Download or read book E. coli Plasmid Vectors written by Nicola Casali and published by Springer Science & Business Media. This book was released on 2008-02-03 with total page 311 pages. Available in PDF, EPUB and Kindle. Book excerpt: A comprehensive collection of readily reproducible techniques for the manipulation of recombinant plasmids using the bacterial host E. coli. The authors describe proven methods for cloning DNA into plasmid vectors, transforming plasmids into E. coli, and analyzing recombinant clones. They also include protocols for the construction and screening of libraries, as well as specific techniques for specialized cloning vehicles, such as cosmids, bacterial artificial chromosomes, l vectors, and phagemids. Common downstream applications such as mutagenesis of plasmids, recombinant protein expression, and the use of reporter genes, are also described.

Protein Kinase C Protocols

Author : Alexandra C. Newton
Publisher : Springer Science & Business Media
ISBN 13 : 1592593976
Total Pages : 565 pages
Book Rating : 4.5/5 (925 download)

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Book Synopsis Protein Kinase C Protocols by : Alexandra C. Newton

Download or read book Protein Kinase C Protocols written by Alexandra C. Newton and published by Springer Science & Business Media. This book was released on 2008-02-03 with total page 565 pages. Available in PDF, EPUB and Kindle. Book excerpt: Since the discovery that protein kinase C (PKC) transduces the ab- dance of signals that result in phospholipid hydrolysis, this enzyme has been at the forefront of research in signal transduction. Protein Kinase C Protocols covers fundamental methods for studying the structure, function, regulation, subcellular localization, and macromolecular interactions of PKC. Protein Kinase C Protocols is divided into 11 sections representing the major aspects of PKC regulation and function. Part I contains an introduction and a historical perspective on the discovery of PKC by Drs. Yasutomi Nishizuka and Ushio Kikkawa. Part II describes methods to purify PKC. Part III describes the standard methods for measuring PKC activity: its enzymatic activity and its stimulus-dependent translocation from the cytosol to the membrane. Part IV describes methods for measuring the membrane interaction of PKC in vivo and in vitro. Part V provides methodologies and techniques for measuring the ph- phorylation state of PKC, including a protocol for measuring the activity of PKC’s upstream kinase, PDK-1. Novel methods for identifying substrates are described in Part VI. Part VII presents protocols for expressing and analyzing the membrane targeting domains of PKC. Part VIII provides a comprehensive c- pilation of methods used to identify binding partners for PKC. Part IX describes pharmacological probes used to study PKC. The book ends with a presentation of genetic approaches to study PKC (Part X) and a discussion of approaches used to study PKC in disease (Part XI).

p53 Protocols

Author : Sumitra Deb
Publisher : Springer Science & Business Media
ISBN 13 : 1592594085
Total Pages : 282 pages
Book Rating : 4.5/5 (925 download)

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Book Synopsis p53 Protocols by : Sumitra Deb

Download or read book p53 Protocols written by Sumitra Deb and published by Springer Science & Business Media. This book was released on 2008-02-02 with total page 282 pages. Available in PDF, EPUB and Kindle. Book excerpt: Since the discovery of p53 as a tumor suppressor, numerous methods have evolved to reveal the unique structural features and biochemical functions of this protein. Several unique properties of p53 posed a challenge to understa- ing its normal function in the initial phase of its research. The low levels of p53 in normal cells, its stabilization under situations of genotoxic stress, induction of growth arrest, and apoptosis with stabilization of the protein, obstructed the visibility of its normal, unmutated function. The property of p53 that can sense a promoter and transactivate or inhibit is still not well understood. It is still not known whether it is the absence of the protein that causes tumorigenesis, or if its mutants have a dominant role in inducing cancer. p53 Protocols comprises eighteen chapters for the study of the diverse properties of p53 and related proteins. The methods included are invaluable for delineating the function of other proteins that may function as tumor suppr- sors or growth suppressors. The chapters are not presented in any schematic order, for the importance and diversity of the functions of p53 make it imp- sible to organize them suitably. We have made a sincere effort to collect the methods most useful to those investigators working on tumor suppressors or growth suppressors. The purpose of p53 Protocols is not only to provide investigators with methods to analyze similar biochemical functions, but also to familiarize them with the associated problems that arose during the course of investigations.

In Vitro Mutagenesis Protocols

Author : Jeff Braman
Publisher : Springer Science & Business Media
ISBN 13 : 1592591949
Total Pages : 284 pages
Book Rating : 4.5/5 (925 download)

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Book Synopsis In Vitro Mutagenesis Protocols by : Jeff Braman

Download or read book In Vitro Mutagenesis Protocols written by Jeff Braman and published by Springer Science & Business Media. This book was released on 2008-02-05 with total page 284 pages. Available in PDF, EPUB and Kindle. Book excerpt: Hands-on researchers with proven track records describe in stepwise fashion their advanced mutagenesis techniques. The contributors focus on improvements to conventional site-directed mutagenesis, including a chapter on chemical site-directed mutagenesis, PCR-based mutagenesis and the modifications that allow high throughput mutagenesis experiments, and mutagenesis based on gene disruption (both in vitro- and in situ-based). Additional methods are provided for in vitro gene evolution; for gene disruption based on recombination, transposon, and casette mutagenesis; and for facilitating the introduction of multiple mutations. Time-tested and highly practical, the protocols in In Vitro Mutagenesis Protocols, 2nd Edition offer today's molecular biologists reliable and powerful techniques with which to illuminate the proteome.

Comprehensive Biomaterials II

Author : Kevin Healy
Publisher : Elsevier
ISBN 13 : 0081006926
Total Pages : 4865 pages
Book Rating : 4.0/5 (81 download)

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Book Synopsis Comprehensive Biomaterials II by : Kevin Healy

Download or read book Comprehensive Biomaterials II written by Kevin Healy and published by Elsevier. This book was released on 2017-05-18 with total page 4865 pages. Available in PDF, EPUB and Kindle. Book excerpt: Comprehensive Biomaterials II, Second Edition, Seven Volume Set brings together the myriad facets of biomaterials into one expertly-written series of edited volumes. Articles address the current status of nearly all biomaterials in the field, their strengths and weaknesses, their future prospects, appropriate analytical methods and testing, device applications and performance, emerging candidate materials as competitors and disruptive technologies, research and development, regulatory management, commercial aspects, and applications, including medical applications. Detailed coverage is given to both new and emerging areas and the latest research in more traditional areas of the field. Particular attention is given to those areas in which major recent developments have taken place. This new edition, with 75% new or updated articles, will provide biomedical scientists in industry, government, academia, and research organizations with an accurate perspective on the field in a manner that is both accessible and thorough. Reviews the current status of nearly all biomaterials in the field by analyzing their strengths and weaknesses, performance, and future prospects Covers all significant emerging technologies in areas such as 3D printing of tissues, organs and scaffolds, cell encapsulation; multimodal delivery, cancer/vaccine - biomaterial applications, neural interface understanding, materials used for in situ imaging, and infection prevention and treatment Effectively describes the many modern aspects of biomaterials from basic science, to clinical applications

Inflammation Protocols

Author : Paul G. Winyard
Publisher : Springer Science & Business Media
ISBN 13 : 1592593747
Total Pages : 373 pages
Book Rating : 4.5/5 (925 download)

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Book Synopsis Inflammation Protocols by : Paul G. Winyard

Download or read book Inflammation Protocols written by Paul G. Winyard and published by Springer Science & Business Media. This book was released on 2008-02-03 with total page 373 pages. Available in PDF, EPUB and Kindle. Book excerpt: Inflammation has been described as the basis of many pathologies of human disease. When one considers the updated signs of inflammation, they would be vasodilation, cell migration, and, in the case of chronic inflam- tion, cell proliferation, often with an underlying autoimmune basis. Gen- ally, inflammation may be divided into acute, chronic, and autoimmune, - though the editors believe that most, if not all, chronic states are often the result of an autoimmune response to an endogenous antigen. Thus, a proper understanding of the inflammatory basis may provide clues to new therap- tic targets not only in classical inflammatory diseases, but atherosclerosis, cancer, and ischemic heart disease as well. The lack of advances in classical inflammatory diseases, such as rh- matoid arthritis, may in part arise from a failure to classify the disease into different forms. That different forms exist is exemplified in patients with d- fering responses to existing antiinflammatory drugs, ranging from nonresponders to very positive responders for a particular nonsteroidal an- inflammatory drug (NSAID). Though researchers have progressively unr- eled the mechanisms, the story is far from complete. It should also be noted that the inflammatory response is part of the innate immune response, or to use John Hunter’s words in 1795, “inflammation is a salutary response.” That may be applied in particular to the defensive response to invading micro- ganisms.

Cytokine Protocols

Author : Marc De Ley
Publisher : Springer Science & Business Media
ISBN 13 : 1592596673
Total Pages : 248 pages
Book Rating : 4.5/5 (925 download)

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Book Synopsis Cytokine Protocols by : Marc De Ley

Download or read book Cytokine Protocols written by Marc De Ley and published by Springer Science & Business Media. This book was released on 2008-02-03 with total page 248 pages. Available in PDF, EPUB and Kindle. Book excerpt: A collection of biochemical, cellular, and molecular techniques for unraveling and quantifying the events occurring between the initial contact of a cytokine at the membrane receptor and the eventual activation of gene transcription. The techniques used include the generation of transfectants, the immunohistochemical detection of cytokines in tissue sections, and optimized staining for cytoplasmic detection. Highlights include RT-PCR of small amounts of mRNA, in situ hybridization, biosensor analysis, measurement of biological activities and standardization, immunohistochemical and single-cell detection, and receptor isolation, characterization, and crystallization. Enjoy a quick and smooth introduction to the key methods used in cytokine research Use readily reproducible techniques that ensure successful experimental results Employ antisense-RNA, RT-PCR of small amounts of mRNA, and in situ hybridization.