Proteome Research Two Dimensional Gel Electrophoresis And Identification Methods

With the end of the Human Genome Project in sight, the next important step is to determine the function of genes. Proteome Research is an important approach to this study and is the first book to comprehensively cover the application of two-dimensional electrophoresis, the central methodology in proteome research. The state-of-the-art is described in detail and the available detection methods are extensively covered. Sufficient detail is given to allow readers to apply these technologies to their own particular requirements.

With the completion of sequencing projects and the advancement of a- lytical tools for protein identification, proteomics—the study of the expressed part of the genome—has become a major region of the burgeoning field of functional genomics. High-resolution 2-D gels can reveal virtually all p- teins present in a cell or tissue at any given time, including posttranslationally modified proteins. Changes in the expression and structure of most cellular proteins caused by differentiation or external stimuli can be displayed and eventually identified using 2-D protein gels. 2-D Proteome Analysis Protocols covers all aspects of the use of 2-D protein electrophoresis for the analysis of biological problems. The contri- tors include many of the leaders in the fields of biochemistry and analytical chemistry who were instrumental in the development of high-resolution 2-D gels, immobilized pH gradients, computer analysis, and mass spectromet- based protein identification methodologies. This book is intended as a benchtop manual and guide both for novices to 2-D gels and for those aficionados who wish to try the newer techniques. Any group using protein biochemistry—especially in the fields of molecular biology, biochemistry, microbiology, and cell biology—should find this book eminently useful. 2-D Proteome Analysis Protocols takes the researcher through the c- plete process of working with 2-D protein gels from making the protein - tract to finally identifying the proteins of interest. It includes protocols for generating 2-D protein extracts from most of the standard model organisms, including bacteria, yeast, nematode, Drosophila, plants, mouse, and human.

Immobilized pH gradients (IPG) represent the most advanced development of isoelectric focusing (IEF). Originally developed to overcome all the problems of IEF in soluble amphoteric buffers (CA) (such as pH gradient instability, complexation with CA chemicals, unreproducibility of pH gradients, protein precipitation at the pI), it turned out to be an entirely new technique, quite different in principle and operation from conventional IEF. The book is thus meant to bring the reader up to date with this fast developing field. The book is divided into six chapters containing information on: detailed treatment of all the chemistry of the Immobiline chemicals; theory of pH gradient generation (computer simulations, tables with all the possible pH recipes); all analytical aspects of IPGs, including staining techniques, blotting etc.; two-dimensional maps, with a detailed treatise of advantages and limitations; preparative aspects of IPGs, including comparison with other preparative electrophoretic techniques; some examples of applications, including genetic and forensic analysis, blood polymorphism etc.The book is extensive and up-to-date, while also extensively covering the theory. Clearly written, with easily accessible information, the volume is a worthy asset to the Lab. Techniques series.

The second edition of this volume provides a comprehensive update of this key method on gel-based proteomics. Chapters present an introduction into the development of methods on principles of differential protein labeling and two-dimensional gel electrophoresis, techniques on optimized proteomic workflows using advanced mass spectrometry for protein identification, and the application of those methods in basic biological research, pathobiology and applied biomarker discovery. 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. Authoritative and cutting-edge, Difference Gel Electrophoresis: Methods and Protocols, Second Edition aims to ensure successful results in the further study of this vital field.

Two-Dimensional Gel Electrophoresis of Proteins: Methods and Applications reviews current methods and clinical applications of two-dimensional gel electrophoresis of proteins, including the QUEST system, silver staining, and peptide mapping. Two-dimensional gel electrophoresis are applied to the study of diseases ranging from inborn errors of metabolism to human germ-line mutation rates, cancer, and mistranslation in animal and bacterial cells. This volume is organized into three sections encompassing 14 chapters and begins with an overview of the methodology of two-dimensional gel electrophoresis, followed by a discussion of computerized two-dimensional gel electrophoresis, silver staining, immunoblotting, and one- and two-dimensional peptide mapping. In most cases, a step-by-step guide to the techniques is given so that procedures may be easily repeated. A catalog of mouse fibroblast proteins is also given. The chapters that follow focus mainly on applications of two-dimensional gel electrophoresis in areas such as clinical and cancer research, human genetics, protein biosynthesis, and gene expression in plants. The final section presents current protein catalogs of Escherichia coli and human HeLa cells. This book is suitable for young researchers as well as for senior scientists working with a wide variety of problems in molecular and cell biology, basic biochemistry, genetics, and clinical research.

This book explores the current status of proteomics, an exciting new discipline, which is less than 10 years old. This new field has rapidly grown into a major commercial and research enterprise with great prospects for dramatically advancing our knowledge of basic biological and disease processes. The contributors to this book are an international panel of proteomics experts, who review and discuss the current status of specific technologies and approaches. Proteomics represents an exciting new way to pursue biological and biomedical science at an unprecedented pace. Proteomics takes a broad, comprehensive, systematic approach to understanding biology that is generally unbiased and not dependent upon existing knowledge. The major components of proteomics from basic discovery using a range of alternative analytical methods to discovery validation and use for clinical applications are discussed. State-of-the-art protein profiling methods include high resolution two-dimensional gels, two-dimensional differential in-gel electrophoresis, LC-MS and LC-MS/MS using accurate mass tags, and protein identifications of proteins from gels using mass spectrometry methods are discussed in depth. Other chapters describe comprehensive characterization of proteomes using electrophoretic prefractionation and analyses of sub-proteomes based on specific posttranslational modifications including the phospho-proteome, the glyco-proteome, and nitrated proteins. These conventional proteome analysis chapters are complemented by discussion of emerging technologies and approaches such as affinity based biosensor proteomics as well as the use of protein microarrays, microfluidics and nanotechnology. Strategies for improving throughput by automation are also discussed. Additional chapters address the application of current proteome techniques to clinical problems and the availability of protein expression library resources for proteome studies. · Authored by international experts in the field · Covers a wide range of topics including 2-D gels, global proteomics using accurate mass tags, global proteomics using electrophoretic prefractionation, microfluidics, and nanotechnology · Includes state-of-the-art protein profiling methods, and emerging technologies

Proteomics is a multifaceted, interdisciplinary field which studies the complexity and dynamics of proteins in biological systems. It combines powerful separation and analytical technology with advanced informatics to understand the function of proteins in the cell and in the body. This book provides a clear conceptual description of each facet of proteomics, describes recent advances in technology and thinking in each area, and provides details of how these have been applied to a variety of biological problems. It is written by expert practitioners in the field, from industry, research institutions, and the clinic. It provides junior and experienced researchers with an invaluable proteomic reference, and gives fascinating glimpses of the future of this dynamic field.

Introduction to the proteome (K. L. Williams, D. F. Hochstrasser). Two-dimensional electrophoresis: the state of the art and future directions (B. R. Herbert, J.-C. Sanchez, L. Bini). large-scale comparative protein modeling ( M. C. Peitsch, N. Guex); Clinical and biomedical applications of proteomics (D. F. Hochstrasser). Biological applications of proteomics (K. L. Williams, V. Pallini). Conclusions (D. F. Hochstrasser, L. Williams). Index.