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The chemokines family of small proteins are involved in numerous b- logical processes ranging from hematopoiesis, angiogenesis, and basal l- kocyte trafficking to the extravasation and tissue infiltration of leukocytes in response to inflammatory agents, tissue damage, and bacterial or viral infection. Chemokines exert their effects through a family of seven G-protein coupled transmembrane receptors. Worldwide interest in the chemokine field surged dramatically early in 1996, with the finding that certain chemokine receptors were the elusive coreceptors, required along with CD4, for HIV infection. Today, though over 40 human chemokines have been described, the n- ber of chemokine receptors lags behind—only 17 human chemokine receptors have been identified so far. What has emerged over the years is that most chemokine receptors bind several distinct ligands, and indeed the majority of chemokines are able to bind to multiple chemokine receptors, explaining to some extent the apparent disparity in the numbers of chemokines and rec- tors. Yet in spite of the apparent redundancy in chemokine/chemokine rec- tor interactions, it is clear that in vivo, spatial, temporal, and indeed cell- and tissue-specific expression of both chemokines and their receptors are imp- tant factors in determining the precise nature of cellular infiltrates in phy- ological and pathological processes.
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.
Cytokines are pleiotropic regulatory proteins involved in essentially all biological processes and associated with a wide variety of diseases, including inflammatory disorders as well as many types of cancer and leukemia. Knowledge about the quantitative and qualitative nature of cytokine production is critical in the understanding of normal and pathological processes. The cytokine detection in biological and clinical samples faces many challenges including their low abundance, the need to distinguish between active and latent cytokine forms, and the need to measure multiple cytokines in a single assay. This volume will provide a comprehensive collection of classic and cutting-edge methodologies that are currently used to analyze and quantify cytokines and their biological activities in complex biological and clinical samples. The chapters are divided into four main categories. The first group focuses on the immunodetection of released cytokines in tissue culture supernatants, plasma, serum and whole blood samples by immunoassays. These immunoassays measure the total concentrations of released cytokines regardless of their biological activity and include ELISA, flow cytometry, ELISPOT and the antibody-based proximity ligation. The second group will focus on the analysis of biologically active cytokines by bioassays using neutralizing antibodies, chemotaxis assay, cytokine-induced cell degranulation assay, cell proliferation and differentiation, cytokine-induced cytokine production and the radioreceptor cytokine assay. The third group focuses on the analysis of intracellular cytokines by flow cytometry, western blotting and fluorescence and confocal microscopy. In addition, this category includes protocols for quantitative analysis of cytokine gene expression by real time RT-PCR and analysis of the cytokine promoter occupancy by chromatin immunoprecipitation. The fourth group focuses on the recently developed multiplex arrays that can measure multiple cytokines in the same sample at the same time. This group includes quantification of multiple cytokines using cytometric bead arrays, ELISPOT assays, proteomics cytokine evaluation, multiplexed proximity ligation assays for high-throughput cytokine analysis and finally, cytokine gene expression analysis by gene arrays. The protocols will be written by experienced basic and clinical researchers with hands-on knowledge of the described protocols. By covering a broad variety of methods used in cytokine detection and analysis, this book will be of interest not only to biochemists, molecular biologists and immunologists but also to physician-scientists working in the field of cytokine research.
As a scientist with an interest in proteins you will, at some time in your career, isolate an enzyme that turns out to be yellow—or perhaps you already have. Alternatively, you may identify a polypeptide sequence that is related to known flavin-containing proteins. This may, or may not, be your first encounter with flavoproteins. However, even if you are an old hand in the field, you may not have exploited the full range of experimental approaches applicable to the study of flavoproteins. We hope that Flavoprotein Protocols will encourage you to do so. In this volume we have sought to bring together a range of experimental methods of value to researchers with an interest in flavoproteins, whether or not these researchers have experience in this area. A broad range of techniques, from the everyday to the more specialized, is described by scientists who are experts in their fields and who have ext- sive practical experience with flavoproteins. The wide range of approaches, from wet chemistry to dry computation, has, as a consequence, demanded a range of formats. Where appropriate (particularly for analytical methods) the protocol described is laid out in easy-to-follow steps. In other cases (e. g. , the more advanced spectroscopies and computational methods) it is far more apt to describe the general approach and relevance of the methods. We hope this wide-ranging approach will sow the seeds of many future collaborations - tween laboratories and further our knowledge and understanding of how f- voproteins work.
Computers have become an essential component of modern biology. They help to manage the vast and increasing amount of biological data and continue to play an integral role in the discovery of new biological relationships. This in silico approach to biology has helped to reshape the modern biological sciences. With the biological revolution now among us, it is imperative that each scientist develop and hone today’s bioinformatics skills, if only at a rudimentary level. Bioinformatics Methods and Protocols was conceived as part of the Methods in Molecular Biology series to meet this challenge and to provide the experienced user with useful tips and an up-to-date overview of current developments. It builds upon the foundation that was provided in the two-volume set published in 1994 entitled Computer Analysis of Sequence Data. We divided Bioinformatics Methods and Protocols into five parts, including a thorough survey of the basic sequence analysis software packages that are available at most institutions, as well as the design and implemen- tion of an essential introductory Bioinformatics course. In addition, we included sections describing specialized noncommercial software, databases, and other resources available as part of the World Wide Web and a stimul- ing discussion of some of the computational challenges biologists now face and likely future solutions.
Adrenergic receptors are important modulators in the sympathetic c- trol of various metabolic processes in the central and peripheral nervous s- tems. These receptors are localized at multiple sites throughout the central nervous system (CNS) and serve as important regulators of CNS-mediated behavior and neural functions, including mood, memory, neuroendocrine c- trol, and stimulation of autonomic function. Adrenergic Receptor Protocols consists of 35 chapters dealing with va- ous aspects of adrenergic receptor analyses, including the use of genetic, RNA, protein expression, transactivator, second messenger, immunocytochemical, electrophysiological, transgenic, and in situ hybridization approaches. This volume details the use of various methods to examine the adrenergic receptor system, using aspects of the genetic flow of information as a guide (DNA? RNA ? transactivator ? protein expression ? second messenger analyses ? cellular analyses ? transgenic whole animal approaches). Adrenergic Receptor Protocols displays step-by-step methods for s- cessful replication of experimental procedures, and would be useful for both experienced investigators and newcomers in the field, including those beg- ning graduate study or undergoing postdoctoral training. The Notes section contained in each chapter provides valuable troubleshooting guides to help develop working protocols for your laboratory. With Adrenergic Receptor Protocols, it has been my intent to develop a comprehensive collection of modern molecular methods for analyzing adrenergic receptors. I would like to thank the many chapter authors for their contributions.
A step-by-step guide to commonly used procedures, Methods in Cellular Immunology addresses both human and murine models, in addition to such topics as PCR and apoptosis. The basic format of the original version has been maintained, and the goal remains the same: to make it a useful and easy-to-use tool for investigators employing cellular immunolog
Mycotoxins produced by molds are common contaminants of many important crops, including wheat, corn, rice, and peanuts. Some mycotoxins are found in fruits and vegetables. These contaminants have a broad range of toxic effects, including carcinogenicity, neurotoxicity, and reproductive and developmental toxicity. The occurrence of mycotoxins in foods is an unavoidable worldwide problem. About 80 countries have imposed regulatory limits to minimize human and animal exposure to mycotoxins. Regulatory limits, including international standards, have tremendous economic impact and must be developed using science-based risk assessments. The purpose of Mycotoxin Protocols is to provide the scientific and technological basis for analytical methods for use in obtaining the exposure data needed for risk assessments. Mycotoxin Protocols is divided into four sections, which are interc- nected. The first section: Chapters 1–5 describe the general techniques for mycotoxin analysis with emphasis on the importance of method validation based on statistical parameters; sampling procedures for collecting a sample as representative as possible of a bulk lot; the isolation of mycotoxins for use as analytical standards or for toxicological studies; the evaluation of purity and preparation of standards; and the detection and identification of impu- ties in isolated mycotoxins. Sections 2–4: Chapters 6–19 describe the most current chromatographic and immunochemical methods for studies on the major mycotoxins.
Established investigators from around the world describe in step-by-step detail their best techniques for the study of plant hormones and their regulatory activities. These state-of-the-art methods include contemporary approaches to identifying the biosynthetic pathways of plant hormones, monitoring their levels, characterizing the receptors with which they interact, and analyzing the signaling systems by which they exert their effects. Comprehensive and fully detailed for reproducible laboratory success, Plant Hormone Protocols offers plant biologists an indispensable compendium of today's most powerful methods and strategies to studying plant hormones, their regulation, and their activities.
The observation that neuropeptide Y (NPY) is the most abundant peptide present in the mammalian nervous system and the finding that it elicits the most powerful orexigenic signal have led to active investigations of the properties of the NPY family of hormones, including peptide YY (PYY) and pancreatic polypeptide (PP). Nearly two decades of research have led to the identification of several NPY receptor subtypes and the development of useful receptor selective ligands. Moreover, these investigations have imp- cated NPY in the pathophysiology of a number of diseases, including feeding disorders, seizures, memory loss, anxiety, depression, and heart failure. Vigorous efforts are therefore continuing, not only to understand the bioche- cal aspects of NPY actions, but also toward developing NPY-based treatments for a variety of disorders. To facilitate these efforts, it was decided to produce the first handbook on NPY research techniques as part of the Methods in Molecular Biology Series. In compiling Neuropeptide Y Protocols, I have gathered contributions on techniques considered critical for the advancement of the NPY field from experts in various disciplines. Each chapter starts with a brief introduction, with Materials and Methods sections following. The latter sections are presented in an easy to follow step-by-step format. The last section of the chapter, Notes, highlights pitfalls and the maneuvers employed to overcome them. This information, not usually disseminated in standard research pub- cations, may prove extremely useful for investigators employing these te- niques in NYP research.