Signal transduction is the fundamental mechanism for regulation of cellular activities by environmental cues and regulatory signals, and is particularly important for plants, whose survival requires proper physiological and developmental responses to the environmental changes. Much progress has been made recently in the plant signal transduction research field thanks to the development of diverse techniques which are covered in Plant Signalling Networks: Methods and Protocols. These include advanced research methods such as proteomics and mass spectrometry methods for studying protein modification, biochemical and cell biological tools for studying protein-protein interactions, genomic techniques for dissecting protein-DNA interaction and transcription networks, and computation methods that integrate molecular network into plant developmental processes. Written in the successful Methods in Molecular Biology (TM) 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. Plant Signalling Networks: Methods and Protocols presents well-honed methodologies for a wide range of research approaches including genetics, proteomics, biochemical, cell biological, and computational approaches, and seeks to serve both professionals and novices with a comprehensive understanding of complex signaling networks in plants.

G protein-coupled receptors (GPCRs) are heptahelical transmembrane receptors that convert extra-cellular stimuli into intra-cellular signaling, and ultimately into biological responses. Since GPCRs are natural targets for approximately 40% of all modern medicines, it is not surprising that they have been the subject of intense research. Notwithstanding the amount of data generated over the years, discovering ligands of these receptors with optimal therapeutic properties is not straightforward and has certainly been hampered for years by the lack of high-resolution structural information about these receptors. Luckily, there has been a steady increase of high-resolution crystal structures of these receptors since 2007, and this information, integrated with dynamic inferences from computational and experimental methods, holds great potential for the discovery of new, improved drugs. This book, which provides, for the first time, state-of-the-art views on modeling and simulation of GPCRs, is divided into 4 parts. In the first part, the impact of currently available GPCR crystal structures on structural modeling is discussed extensively as are critical insights from simulations in the second part of the book. The third part reports recent progress in rational ligand discovery and mathematical modeling, whereas the fourth part provides an overview of bioinformatics tools and resources that are available for GPCRs.

Protein expression in a heterologous host is a cornerstone of biomedical research and of the biotechnology industry. Despite the advanced state of protein expression technology improvements are still needed. For example, membrane proteins constitute a significant percentage of the total cellular proteins but as a class are very difficult to overexpress, especially in a heterologous host. The ideal host would have the ability to express any protein, with relevant post-translational modifications, and be as easy to work with as E. coli. In Heterologous Gene Expression in E. coli: Methods and Protocols, expert scientists intimately familiar with the relevant techniques offer chapters that greatly expand the utility of this expression host. The contributions in this detailed volume describe methods, for example, to successfully express proteins in E. coli that would otherwise form aggregates in this host, to add post-translational modifications, to incorporate non-standard amino acid residues or moieties into E. coli expressed proteins, to identify binding partners, and to express membrane proteins. Written in the highly successful Methods in Molecular Biology (TM) format, chapters include introductions to their respective subjects, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls. Practical and cutting-edge, Heterologous Gene Expression in E. coli: Methods and Protocols seeks to familiarize the researcher with the myriad of E. coli expression strains available and move E. coli closer to that ideal of the perfect host.

The field of protein NMR spectroscopy has rapidly expanded into new areas of biochemistry, molecular biology and cell biology research that were impossible to study as recently as ten years ago. This third edition of Protein NMR Techniques, expands upon the previous editions with current, detailed authoritative but down-to-earth descriptions of new methodologies. These include techniques for NMR sample preparation, solution and solid state NMR methodologies and data processing. Written in the highly successful Methods in Molecular Biology (TM) series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and key tips on troubleshooting and avoiding known pitfalls. Authoritative and practical, Protein NMR Techniques,Third Edition, seeks to aid scientists in understanding the latest innovations in the field of protein NMR.

Cellular adhesion is a fundamental process that influences numerous biological activities such as morphogenesis, cell motility and division, as well as signalling. In addition, adhesion is a process important not only in normal physiology and development, but also in disease states such as tumourigenesis, cardiovascular disease, inflammation and infection. There are a plethora of proteins involved in adhesion-related events with a huge diversity in function. As a result, a wide variety of techniques exist to study adhesion related proteins and processes. In Adhesion Protein Protocols, Third Edition, chapters cover techniques to gain insight into the complex and incompletely understood processes that are involved in cellular adhesion. 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, Adhesion Protein Protocols, Third Edition will be useful for both those new to the field of adhesion protein research as well as the more experienced scientist.

Proteostasis is central to the development of various human diseases caused due to excessive protein misfolding and the disregulation of the protein quality control system. In this book, respected researchers from many leading institutions contribute their insights on proteostasis maintenance. The coverage mainly focuses on the basics of maintaining proteostasis, the consequences of proteostatic system failure, and how chaperone systems constantly maintain proteostasis. In addition, the book presents in detail different treatment strategies for diseases caused by proteostatic system failure, as well as the inhibition of proteostatic failure using small molecule compounds. It examines advances in the modulation of proteopathies, providing a comprehensive source of key mechanistic insights on these diseases. As such, the book offers a valuable resource for beginners and more experienced investigators alike who are looking for detailed and reliable information on protein homeostasis, the diseases that can develop due to related imbalances and the essential role of molecular and chemical chaperones.

The discovery of microRNAs and its role as gene expression regulators in human carcinogenesis represents one of the most important scientific achievements of the last decade. More recently, other non-coding RNAs have been discovered and its implications in cancer are emerging as well, suggesting a broader than anticipated involvement of the non-coding genome in cancer. Moreover, completely new and unexpected functions for microRNAs are being revealed, leading to the identification of new anticancer molecular targets. This book represents a comprehensive guide on non-coding RNAs and cancer, spanning from its role as cancer biomarkers, to providing the most useful bioinformatic tools, to presenting some of the most relevant discoveries, which indicates how these fascinating molecules act as fine orchestrators of cancer biology.

The genomes of cellular organisms are organized as double-stranded DNA, a structure that must be unwound to provide DNA replication, recombination, and repair machinery access to genomic information. However, DNA unwinding comes with inherent risks to genome stability. To help mediate these risks, bacterial, archael, and eukaryotic cells have evolved protective ssDNA-binding proteins (SSBs) that bind ssDNA with high affinity and specificity. SSBs also aid genome metabolic processes through direct interactions with key proteins in genome maintenance enzymes. Single-Stranded DNA Binding Proteins: Methods and Protocols assembles methods developed for examining the fundamental properties of SSBs and for exploiting the biochemical functions of SSBs for their use as in vitro and in vivo reagents. Clearly and concisely organized, the volume opens with an introduction to the structures and functions of SSBs, followed protocols for studying SSB/DNA complexes, methods for studying SSB/heterologous protein complexes, protocols for interrogating post-translational modifications of SSBs, and concludes with uses of fluorescently-labeled SSBs for in vitro and in vivo studies of genome maintenance processes. Written in the successful Methods in Molecular Biology (TM) 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, Single-Stranded DNA Binding Proteins: Methods and Protocols provides a rich introduction for investigators who are interested in this fascinating family of DNA-binding proteins.

Now recognized as a reservoir for growth factors and cytokines modulating cell activation status and turnover, proteoglycans have stimulated great amount of interest and research. In Proteoglycans: Methods and Protocols, experts in the field examine issues of basic concepts and up-to-date analysis methods for proteoglycan and glycosaminoglycan (GAG) at the protein and saccharide levels. The volume continues with the multifunctional aspect of proteoglycans, highlighted through three relevant examples of proteoglycans with highly different strucutures: serglycin, aggrecan, and heparin sulphate proteoglycans. The final chapter describes proteoglycan involvement in the pathogenesis of various disorders and their potential therapeutic value in osteo-articular diseases. Written in the highly successful Methods in Molecular Biology (TM) 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. Authoritative and accessible, Proteoglycans: Methods and Protocols is an ideal guide for scientists attempting to pursue further research in this vital and complex area of study.

Thousands of proteins have been identified to be acetylated. Immense research power has been dedicated to experiments to solve the biological implications of each and every protein acetylation. Two particular sites of protein acetylation have been described intensively: the N-terminal methionine residue of a nascent protein and lysine residues within a protein. In Protein Acetylation: Methods and Protocols, expert researchers in the field detail many of the methods which are now commonly used to study protein acetylation. These include methods and techniques for identification of protein acetylation, column- and gel electrophoresis-based approaches, computationally prediction, and the biological response to protein acetylation. Written in the highly successful Methods in Molecular Biology (TM) series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and key tips on troubleshooting and avoiding known pitfalls. Authoritative and practical, Protein Acetylation: Methods and Protocols seeks to aid scientists in the further study of the technical aspects involved in understanding protein acetylation.

Display technologies have become a very powerful way of generating therapeutic lead molecules and specific reagents for increasing our understanding of biology; however, despite being first described shortly after phage display, the use of ribosome display and related methods have been much less widespread. Since this is in part due to the complexity of the methods, Ribosome Display and Related Technologies: Methods and Protocols seeks to extend their use by collecting expert contributions describing these detailed protocols. The protocols described range from well-established methods that have been used for a decade to generate high affinity antibodies, which are already in the clinic, to methods that are in their early stages of application such as display of peptides incorporating non-canonical amino acids. Written in the highly successful Methods in Molecular Biology (TM) 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. Invaluable and easy to use, Ribosome Display and Related Technologies: Methods and Protocols will be of great benefit to those with general molecular biology or protein engineering experience who wish to select peptides or proteins by display, those with phage display experience who would benefit from the application of ribosome display, as well as those with some ribosome display experience who would like to expand the range of applications to which they are applying the technology.

Proteins are the functional units of the cellular machinery and they provide significant information regarding the molecular basis of health and disease. Therefore, techniques to separate and isolate the various proteins are critical to studying and understanding their functional characteristics. One of the widely used techniques for this purpose is electrophoresis. In Protein Electrophoresis: Methods and Protocols, contributions from experts in the field have been collected in order to provide practical guidelines to this complex study. Each chapter outlines a specific electrophoretic variant in detail so that laboratory scientists may perform a technique new to their lab without difficulty. Written in the successful Methods in Molecular Biology (TM) 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 accessible, Protein Electrophoresis: Methods and Protocols seeks to serve laboratory scientists with well-honed, detailed methodologies in an effort to further our knowledge of this essential field.

This first of two volumes provides a general overview of the genetics, structure, mechanism and regulation of the Ras superfamily proteins and describes in detail the signaling pathways and processes regulated by specific members of this family. The focus of this first volume is on the Rho and Ras subfamily of small G proteins. Renowned scientists provide insights into the biochemistry of the classical and non-classical small G-protein family members, their spatio-temporal regulation, their effectors and their roles in health and disease. Together with Volume 2, this book provides a comprehensive and state-of-the-art work on small G-proteins (GTPases). It is intended for graduates and professors in biochemistry and cell biology already working on small G-proteins (small GTPases), but also offers an extremely valuable resource for those readers who are new to the field.

Progress in functional proteomics has been limited for a long time, partially caused by limitations in assay sensitivity and sample capacity; however, protein microarrays have the ability to overcome these limitations so that a highly parallel analysis of hundreds of proteins in thousands of samples is attainable. In Protein Microarrays: Methods and Protocols, expert researchers in the field present an up-to-date collection of robust strategies in the field of protein microarrays and summarize recent advantages in the field of printing technologies, the development of suitable surface materials, as well as detection and quantification technologies. Written in the highly successful Methods in Molecular Biology (TM) series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and key notes on troubleshooting and avoiding known pitfalls. Comprehensive and cutting-edge, Protein Microarrays: Methods and Protocols aims to stimulate the application and further advancement of this powerful technology in labs worldwide.

Membrane proteins play key roles in numerous cellular processes, in particular mediating cell-to-cell communication and signaling events that lead to a multitude of biological effects. Membrane proteins have also been implicated in many critical diseases such as atherosclerosis, hypertension, diabetes and cancer. In Membrane Protein Structure Predictions Methods: Methods and Protocols, expert researcher in the field detail the advances in both experimental and computational approaches of the structure, dynamics and interactions of membrane proteins dividing the volume into two sections. The first section details the procedures used for measurements of structure and dynamics of membrane proteins. While the second section contains a survey of the computational methods that have played a critical role in membrane protein structure prediction as well as in providing atomic level insight into the mechanism of the dynamics of membrane receptors. Written in the highly successful Methods in Molecular Biology (TM) series format, the chapters include the kind of detailed description and implementation advice that is crucial for getting optimal results in the laboratory. Thorough and intuitive, Membrane Protein Structure Predicitons: Methods and Protocols seeks to aid scientists in the further study of membrane protein structure and function.

This excellent book covers wide-ranging topics in interdisciplinary microbiology, addressing various research aspects and highlighting advanced discoveries and innovations. It presents the fascinating topic of modern biotechnology, including agricultural microbiology, microalgae biotechnology, bio-energy, bioinformatics and metagenomics, environmental microbiology, enzyme technology and marine biology. It presents the most up-to-date areas of microbiology with an emphasis on shedding light on biotechnological advancements and integrating these interdisciplinary microbiology research topics into other biotechnology sub-disciplines. The book raises awareness of the industrial relevance of microbiology, which is key component of this unique collection. The topics include production of antioxidant-glutathione, enzyme-engineering methods, probiotic microbiology and features of microbial xylanases. It also covers some other remarkable aspects of microbiology, like potential health hazards in recreational water and fullerene nanocomposites, which are vital for biotechnological interventions. This book will be valuable resource for senior undergraduate and graduate students, researchers and other interested professionals or groups working in the interdisciplinary areas of microbiology and biotechnology.

The liver is responsible for a wide range of critical functions essential to life, and is composed of several different cell types. In Liver Proteomics: Methods and Protocols, expert researchers in the field detail many of the methods that are used to study the live. These methods include the most up-to-date strategies being used to characterize the liver proteome at the global, cellular, subcellular, post translational and functional level.Written in the highly successful Methods in Molecular Biology (TM) series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and key tips on troubleshooting and avoiding known pitfalls. Authoritative and practical, Liver Proteomics: Methods and Protocols seeks to aid scientists in the further study of this crucially important organ.

Exploring the 2-D gel mapping field, the chapters in this book are separated into four different categories: Part I talks about 2-D maps reproducibility and maps modeling; Part II describes the image analysis tools that provide spot volume datasets; Part III is about the statistical methods applied to spot volume datasets to identify candidate biomarkers; and Part IV discusses differential analysis from direct image analysis tools. 2-D PAGE Map Analysis: Methods and Protocols provides a unique approach to 2-D gel mapping, in that it helps users avoid drawbacks due to ignorance of the basic theoretical mechanisms underlying the technique, including data handling and proper tools for spot analysis. Written in the highly successful Methods in Molecular Biology series format, chapters include introductions to their respective topics, lists of the necessary materials, step-by-step, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls. Cutting-edge and thorough, 2-D PAGE Map Analysis: Methods and Protocols, is a useful resource for any scientist or researcher, with a mathematical background, who is interested in 2-D gel mapping.

The study of carbonic anhydrase has spanned multiple generations of scientists. Carbonic anhydrase was first discovered in 1932 by Meldrum and Roughton. Inhibition by sulfanilamide was shown in 1940 by Mann and Keilin. Even Hans Krebs contributed to early studies with a paper in 1948 showing the relationship of 25 different sulfonamides to CA inhibition. It was he who pointed out the importance of both the charged and uncharged character of these compounds for physiological experiments. The field of study that focuses on carbonic anhydrase (CA) has exploded in recent years with the identification of new families and isoforms. The CAs are metalloenzymes which are comprised of 5 structurally different families: the alpha, beta, gamma, and delta, and epsilon classes. The alpha class is found primarily in animals with several isoforms associated with human disease. The beta CAs are expressed primarily in plants and are the most divergent. The gamma CAs are the most ancient. These are structurally related to the beta CAs, but have a mechanism more similar to the alpha CAs. The delta CAs are found in marine algae and diflagellates. The epsilon class is found in prokaryotes in which it is part of the carboxysome shell perhaps supplying RuBisCO with CO2 for carbon fixation. With the excitement surrounding the discovery of disease-related CAs, scientists have redoubled their efforts to better understand structure-function relationships, to design high affinity, isotype-specific inhibitors, and to delineate signaling systems that play regulatory roles over expression and activity. We have designed the book to cover basic information of mechanism, structure, and function of the CA families. The authors included in this book bring to light the newest data with regard to the role of CA in physiology and pathology, across phylums, and in unique environmental niches.

The second edition of p53 Protocols expands upon the first edition with progress in p53 research since the publication of the first edition. In p53 Protocols, Second Edition expert researchers in the field detail many of the methods that address the challenging questions of the p53 field. These methods include the identification of the target genes and binding partners of gain of function p53 mutants, methods to determine stress response, autophagy or senescence induced by p53, cell cycle analysis, analysis of different phases of DNA replication modified by p53, and generation of pluripotent stem cells. Written in the highly successful Methods in Molecular Biology (TM) series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and key tips on troubleshooting and avoiding known pitfalls. Authoritative and practical, p53 Protocols, Second Edition seeks to aid scientists in the further study into p53 and other tumor suppressors.

This book covers the latest developments in the physical biology of proteins and peptides. Key insights into microscopic and macroscopic approaches to describe biologically relevant macromolecules and their interactions are provided. This book also covers a wide range of tools, including theoretical methods as statistical mechanics, normal mode analysis, kinetic theory and stochastic processes, and all-atom and coarse-grained molecular dynamics simulations. New experimental techniques are also discussed, particularly related to amiloidogenic peptides and their mutations. This is an excellent book for molecular biologists, physicists, computational scientists, and chemists. It covers cutting-edge research in this exciting, interdisciplinary research field. This book also: Discusses the latest developments in the physical biology of proteins, peptides and enzymes covering theoretical, computational, and experimental approaches Broadens readers' understanding on the r ole of intra- and inter-molecular interactions as a fundamental cornerstone of macroscopic biological properties of macromolecules Provides a wide and useful perspective on different aspects of the physics, biology, and chemistry of proteins and peptides suitable for interdisciplinary research.

Since the first edition of Protein Nanotechnology Protocols Instruments and Applications the intersection of protein science and nanotechnology has become an exciting frontier in interdisciplinary sciences. The second edition of Protein Nanotechnology Protocols Instruments and Applications expands upon the previous editions with current, detailed chapters that provide examples of proteins which are now being harnessed for a wide range of applications, some more developed than others. This book also delves into engineering proteins and an overview of the sorts of tools that are now readily available to manipulate the structure and function of proteins, both rationally and using methods inspired by evolution. Written in the highly successful Methods in Molecular Biology (TM) series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and key tips on troubleshooting and avoiding known pitfalls. Authoritative and practical, Protein Nanotechnology Protocols Instruments and Applications, Second Edition seeks to provide an overview of this multi-faceted field and a useful guide to those who wish to contribute to it.

Among the many types of DNA binding domains, C2H2 zinc finger proteins (ZFPs) have proven to be the most malleable for creating custom DNA-binding proteins. In Engineered Zinc Finger Proteins: Methods and Protocols, expert researchers from some of the most active laboratories in this field present detailed methods, guidance, and perspectives. The volume contains sections covering the engineering of ZFPs, methods for the creation, evaluation, and delivery of artificial transcription factors (ATFs), methods for the creation and evaluation of zinc finger nucleases (ZFNs), and a collection of the several applications and assays beyond ATFs and ZFNs, including zinc finger transposases and ChIP-seq methodology amongst other subjects. Written in the highly successful Methods in Molecular Biology (TM) series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and notes on troubleshooting and avoiding known pitfalls. Comprehensive and cutting-edge, Engineered Zinc Finger Proteins: Methods and Protocols aims to aid both seasoned practitioners and new investigators with its vital methods and insights as they seek to create the next generation of engineered ZFPs and applications.

Systems Biology represents a new paradigm aiming at a whole-organism-level understanding of biological phenomena, emphasizing interconnections and functional interrelationships rather than component parts. The study of network properties, and how they control and regulate behavior from the cellular to organism level, constitutes a main focus of Systems Biology. This book addresses from a novel perspective a major unsolved biological problem: understanding how a cell works and what goes wrong in pathology. The task undertaken by the authors is in equal parts conceptual and methodological, integrative and analytical, experimental and theoretical, qualitative and quantitative, didactic and comprehensive. Essentially, they unravel the spatio-temporal unfolding of interacting mass-energy and information networks at the cellular and organ levels, as well as its modulation through activation or repression by signaling networks to produce a certain phenotype or (patho)physiological response. Starting with the historical roots, in thirteen chapters this work explores the Systems Biology of signaling networks, cellular structures and fluxes, organ and microorganism functions. In doing so, it establishes the basis of a 21st century approach to biological complexity.

Providing current diverse approaches and techniques used to study the immunoproteome, Immunoproteomics: Methods and Protocols collects chapters from key researchers that deliver information to be used in diagnostics, disease progression, and vaccine correlates of protection analysis, to name but a few. This detailed volume includes techniques used for the study of the antibody targets of bacterial pathogens, viruses, and cancer, mass spectrometry-based approaches to characterize T-cell epitopes, chapters on detection and relative quantification of cytokines in serum, as well as in silico prediction of epitopes using sequence-based or modeling approaches. 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. Practical and thorough, Immunoproteomics: Methods and Protocols aids researchers in transferring these techniques to their own laboratories in addition to providing a reference to guide researchers toward appropriate techniques.