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In the last 10 years researchers have firmly established key roles for R- related GTPases in almost every aspect of cell biology. In the 1980s the pro- oncogene Ras itself was the focus of interest, though in the 1990s this shifted to the increasing variety of Ras-related proteins. In this new decade much yet needs to be done to establish the role for all the small GTPases now uncovered by the human genome project. In particular, these GTPases need to be und- stood in the appropriate biochemical and cellular contexts. In the process of trying to uncover the role of these versatile proteins, a variety of novel te- niques and methodologies has been developed. These now enable investi- tors to move easily within a diversity of fields ranging from structural studies to real-time in vivo analysis of a GTPase. In recognition of the need for access to key background methodologies, GTPase Protocols: The Ras Superfamily is devoted to techniques that are pr- ently widely used and that will continue to be the standard for researchers worldwide. Each chapter is aimed at supplying detailed methodologies to allow reproduction in any laboratory, while also providing the general pr- ciples on which the methods are based. Some of the techniques grouped in the first section apply broadly to small GTPases, whereas others in Part II are more applicable within each GTPase subfamily.
As our understanding of the biological sciences expands, the bou- aries between traditional disciplines tend to blur at the edges. Physio- gists and pharmacologists, for instance, now need to embrace techniques that until recently were the strict preserves of biochemists and mole- lar biologists. However, the acquisition of new technologies can be a time-consuming and frustrating business, and unless an expert is on hand to give instruction, precious hours can be spent poring over half-described Methods sections with no guarantee of eventual success. The aim of Signal Transduction Protocols has been to get experts with "hands-on" experience in particular techniques to give detailed accounts of experimental protocols in a recipe-type format, which we hope will circumvent the problems of ambiguity often encountered when reading the literature. The techniques described in Signal Transduction Protocols are those that we think will be most useful in addressing questions in the area of receptor-mediated cell signaling, with particular regard to those receptors that are part of the G-protein-linked superfamily. To keep it to a manageable size, we have omitted any reference to electrophysi- ogy and have instead concentrated on more biochemical approaches.
This book provides a concise set of protocols for assessing basic neutrophil functions, investigating specialized areas in neutrophil research, and completing step-by-step diagnostic assays of common neutrophil disorders. Each of the protocols is written by leading researchers in the field and includes hints for success, as well as guidance for troubleshooting. Scientists and clinicians will find this collection an invaluable aid.
There are a number of outstanding volumes that provide a comprehensive overview of bioconjugation techniques. However, many of the conventional approaches to the synthesis of chemically modified protein conjugates lack efficient means to control the stoichiometry of conjugation, as well as the s- cific site of attachment of the conjugated moiety. Moreover, the recent dev- opments in microarray technologies as well as in nanobiotechnology—a novel field of research rapidly evolving at the crossroads of physics, chemistry, b- technology, and materials science—call for a summary of modern bioconjugation strategies to overcome the limitations of the classical approaches. Bioconjugation Protocols: Methods and Strategies is intended to provide an update of many of the classic techniques and also to introduce and summarize newer approaches that go beyond the pure biomedical applications of bioconjugation. The purpose of Bioconjugation Protocols: Methods and Str- egies is therefore to provide instruction and inspiration for all those scientists confronting the challenges of semisynthesizing functional biomolecular reagents for a wide variety of applications ranging from novel biomedical diagnostics, to therapeutics, to biomaterials. Part I contains seven protocols for the preparation of protein conjugates.
Integrated Methods in Protein Biochemistry: Part A, Volume 677, the latest release in the Methods in Enzymology series, highlights new advances in the field with this new volume presenting interesting chapters on topics such as DNA and protein engineering to create protein bioswitches with new functions, Interaction and cross-talk of prelamin A with integral membrane zinc metalloproteases, An experimental protocol to study lipid transfer proteins, Synthesis of small heat shock proteins, Druggable p-p interacting sites for Co-chaperone DNAJA1 and its partner proteins, An experimental protocol for glycoconjugate analysis, Methods for proximity-based biotinylation combined with Mass Spectrometry, and more. Additional chapters cover Synthetic antibody fragments as conformational sensors of protein activation and trafficking, Expression, purification, functional analysis and crystallization of Rag GTPase, Purification of bacterial transcription elongation complexes by photoreversible immobilization, Inhibition of c-Myc-MAX heterodimerization, Fluorogenic RNA aptamers to probe transcription by multi-subunit RNA polymerases, and much more. - Provides the authority and expertise of leading contributors from an international board of authors - Presents the latest release in the Methods in Enzymology series - Updated release includes the latest information on Integrated Methods in Protein Biochemistry
The previous edition of Transmembrane Signaling Protocols was published in 1998. Since then the human genome has been completely sequenced and new methods have been developed for the use of microarrays and proteomics to analyze global changes in gene expression and protein profiles. These advances have increased our ability to understand transmembrane signaling processes in much greater detail. They have also simultaneously enhanced our ability to determine the role of a large number of newly identified molecules in signaling events. In addition, novel video microscopy methods have been developed to image transmembrane signaling events in live cells in real time. In view of these major advances, it is time to update the previous edition. Because of the success of that volume, we have chosen to keep the essential character of the book intact. Introductory chapters from experts have been included to provide overall perspective and an overview of recent advances in signal transduction pathways. The individual chapters now include comp- hensive detailed methods, studies in genetically tractable systems, fluorescence microscopy in live single cells, ex vivo analysis of primary cells from tra- genic mice, as well as genomic and proteomic approaches to the analysis of transmembrane signaling events. We would like to express our deep gratitude to the coauthors of this publi- tion. We hope that Transmembrane Signaling Protocols, Second Edition will serve as a valuable resource for future progress in the study of signal transd- tion pathways.
This second edition of Receptor Signal Transduction Protocols not only has a new editor, but also a greater focus on G-protein-coupled receptors, their properties per se, and their coupling to immediate downstream binding partners—principally, although not exclusively, the heterotrimeric G-proteins. The new edition combines updates of key chapters from the first edition, as well as a large number of new contributions covering key methodologies that have emerged, or been extended to receptor/G-protein research, in the past 5–6 years. In common with many fields, the range of methods used to assess the first steps in signal transduction are continually expanding and methods that might have been considered too specialized five years ago are now sufficiently routine to be included here. Unlike many research areas, where off-the-shelf kits have made research basically foolproof, signal transduction research still requires considerable expertise, and the methods included here are provided by internationally recognized experts in their fields who have many years of experience using the methods they describe. This not only allows each chapter to impart a clear description of the method, but also to furnish invaluable troubleshooting advice for when things do not go entirely according to plan. Once again we would like to thank the Series Editor, John Walker, for the invitation to compile this second edition, and to express our gratitude to all of the authors who have enthusiastically agreed to provide the uniformly excellent contributions.
In 1995, Signal Transduction Protocols, edited by David A. Kendall and Stephen J. Hill, was published in the Methods in Molecular Biology series. This second edition represents an update to that previous work with an emp- sis on new methodologies that have developed in the last few years. The goal, then and now, is to provide procedures written by experts with first-hand ex- rience in a detail that goes far beyond what is generally encountered in the “methods” section of most journals and thus actually permits a particular p- cedure to be replicated. In addition, we have had as a secondary goal the id- tification of protocols for the assay of general classes of signal transduction components that, ideally, can be adapted to the assay of any member of that class. The ability to do this has resulted in large part from the use of affini- based assays, the ease with which specific proteins can be specifically tagged, and an explosion in the availability of highly specific antibodies from comm- cial sources, especially antibodies raised against signaling proteins of human origin. The number of available approaches is, fortunately for those working in signaling research, far too great to fit within the confines of this volume, so hard choices as to what to include had to be made.
This collection of practical, cutting-edge techniques for the study of cell signaling provides detailed, step-by-step instructions, helpful notes, and troubleshooting tips that make even the most powerful of the newest techniques readily reproducible. The protocols presented include the use of peptide libraries to study transmembrane signaling; the use of single-cell assays to analyze signal transduction pathways; the reconstitution of signaling complexes; methods for analyzing protein-protein interactions, and more. Introductory reviews explain the basic theory and enable researchers new to the area to rapidly gain understanding, as well as command of the practical knowledge and expertise afforded by the protocols. Transmembrane Signaling Protocols makes available to all researchers the many state-of-the-art techniques that have recently led to landmark discoveries in transmembrane signaling.
Mitogen-activated protein kinase (MAPK) signaling cascades are a group of protein kinases that play a central role in the intracellular transmission of extracellular signals. These cascades operate as major lines of communication within a complicated signaling network that regulates many cellular processes, including proliferation, differentiation, development, stress response, and apoptosis. More than 15,000 papers on MAPKs have been published over the past few years, with the number of publications increasing each year. More and more laboratories embark on the study of MAPK cascades in many d- tinct cellular systems and in particular their role in disease. Future challenges in the study of MAPK cascades remain in understa- ing the role of the various components and isoforms of the cascades in the multiple critical functions that they regulate in the whole organism, as well as the diseases caused by their malfunction. Data from gene-disrupted mice s- gest that inhibition of the MAPK cascades may have serious consequences on the development and growth of the animals. For example, targeted deletion of MEK1 is lethal, owing to developmental problems of placental vasculature and abnormal fibroblast migration. This lethality occurs in spite of the normal expression of MEK2, indicating that although the two MEK isoforms are apparently similar, they do have distinct functions, at least during embryog- esis. The ERK cascade was also shown to play a central role in brain function and in learning and memory.