Springer Lab Manuals – serie

The Maize Handbook represents the collective efforts of the maize research community to enumerate the key steps of standard procedures and to disseminate these protocols for the common good. Although the material in this volume is drawn from experience with maize, many of the procedures, protocols, and descriptions are applicable to other higher plants, particularly to other grasses. The power and resolution of experiments with maize depend on the wide range of specialized genetic techniques and marked stocks; these materials are available today as the culmination of nearly 100 years of genetic research. A major goal of this volume is to introduce this genetical legacy and to highlight current stock construction programs that will soon benefit our work, e. g. high-density RFLP maps, deletion stocks, etc. Both stock construction and maintenance are relatively straightforward in maize as a result of the ease of crossing and the longevity of stored seeds. Crossing is facilitated by the separate staminate (tassel) and pistillate (ear) flowers, a feature almost unique to maize. On the other hand, many of the genetic methodologies utilized with maize, including the precision of record keeping, can be adapted to other plants. Facile communication and a spirit of co-operation have characterized the maize genetics community since its earliest days. Starting in the 1930s, institutions such as annual Maize Genetics Cooperation Newsletter, the Maize Genetics Stock Center, and the annual maize genetics meeting provide continuity to the field.

Endothelial cell biology has developed into a vibrant discipline and has become a critical instrument to study several disease processes on the cellular and molecular level. It is now widely recognized that dysfunctions of normal endothelial cell homeostasis are involved in some of the most important human diseases, including ischemic heart diseases, hypertension, atherosclerosis, tumors, diabetes, arthritis, and inflammation. Further, the increasing importance and recognition of the field of vascular biology in general requires in vitro and in vivo techniques in order to address the complex questions.Methods in Endothelial Cell Biology is a comprehensive practical "how-to"-guide summarizing the most relevant established techniques as well as a number of new emerging techniques. Easy-to-follow reliable protocols provide a useful lab bench resource for the experienced researcher and newcomer to the field.

Basic Methods for the Biochemical Lab elucidates proven lab procedures and practical hints for research in analytical and preparative biochemistry, as well as summarizing key data in numerous tables. To further emphasize the practical aspects and to minimize the text, theoretical introductions into the methods are mostly omitted. The chapters cover: quantitative methods (proteins, nucleic acids, phospholipids and carbohydrates), electrophoresis (several polyacrylamide and agarose systems, 2D-PAGE, detection methods and affinity electrophoresis), chromatographic protocols (thin-layer chromatography, GPC, IEC, affinity chromatography, HPLC), immunochemical protocols (hapten-carrier and enzyme conjugation, immunization and antibody purification, immune affinity chromatography, ELISA), centrifugation (differential and density gradient centrifugation for cells and cellular fractions), radioactivity (labeling and counting), buffers (buffer properties and compositions). In additional chapters, tables compile a broad array of useful information, e.g. SI units conversion factor, detergent, protein and nucleotide data, and the basic principles of statistics and enzyme and receptor kinetics are reviewed.This first English-language edition of a successful German-language manual will be a valuable resource for students and experts in biochemistry, biotechnology and biomedical laboratories.

As an intricate association between a fungus and one or more green algae or cyanobacteria, lichens are one of the most successful examples of symbiosis. These fascinating organisms survive extreme desiccation and temperatures. They are adapted to a great variety of habitats, from deserts to intertidal zones, from tropical rain forests to the peaks of the Himalayas and to circumpolar ecosystems. Lichens are extremely efficient accumulators of atmospherically deposited pollutants, and are therefore widely used to monitor environmental pollution. Their wide range of secondary products show pharmaceutically interesting fungicidal, antibacterial and antiviral properties. Lichens are extremely difficult to culture. This manual provides well-tested tissue culture protocols, protocols for studying lichen ultrastructure, (eco)physiology, primary and secondary compounds, and for using lichens as bioindicators.

Proteins interacting with diverse ligands - proteins, peptides or DNA - are the basic principles underlying many biological processes, such as antigen-antibody binding, signal transduction or receptor binding.The technique of oligopeptide synthesis on a cellulose membrane and the subsequent binding assays allow the investigation of protein interactions. A particular advantage of these peptide arrays (SPOT - technology) is the high number of oligopeptide probes that can be tested in parallel. Detailed protocols for peptide synthesis, and the analysis of protein-protein, protein-DNA interactions as well as epitope mapping are presented in this manual. It is ideally suited not only for basic research laboratories but also for diagnostic and therapeutic applications since many diseases are related to dysfunctions in protein recognition and binding.

Most information on yeasts derives from experiments with the conventional yeasts Saccaromyces cerevisiae and Schizossaccharomyces pombe, the complete nuclear and mitochondrial genome of which has also been sequenced. For all other non-conventional yeasts, investigations are in progress and the rapid development of molecular techniques has allowed an insight also into a variety of non-conventional yeasts.In this bench manual, over 70 practical protocols using 15 different non-conventional yeast species and in addition several protocols of general use are described in detail. All of these experiments on the genetics, biochemistry and biotechnology of yeasts have been contributed by renowned laboratories and have been reproduced many times. The reliable protocols are thus ideally suited also for undergraduate and graduate practical courses.

Principles of Peptide Synthesis is a very successful book by one of the pioneers of contemporary bioorganic synthesis. Its first edition proved a valuable source that is kept close to the work bench for frequent consulting. Any researcher planning the formation of the peptide bond, be it for the synthesis of a peptide chain or a cyclic peptide, benefits from the author's experience. The concise and critical treatment of the steps involved: protection, activation and bond formation require careful planning to avoid racemization and undesired side reactions. The second edition has been completely revised and updated. New procedures that have been developed since the first edition was published and that did not fit into the original are added in a separate chapter. This separation of old and new make it possible to assess new ideas and discern novel trends.

During the years 1980-81, as guests of the Deutsches Wollforschungsinstitut in Aachen, Germany, we were working on a small book entitled, "Principles of Peptide Synthesis". In the library of the Institute we noted that the volumes of Houben-Weyl's Handbuch der Organischen Chemie dealing with peptide synthesis were so much in use that they were ready to fall apart because the researchers of the Institute consulted them with amazing regularity. They were looking for references, but even more for experimental details which could be adapted to the particular problem they happened to face. In planning a new synthetic endeavor they tried to lean on the experience of others in analogous situations. This suggested to us that a smaller and hence more tractable book may be needed, a volume which can be kept on or near the bench to make examples of fundamental methods readily available in the laboratory. Such a collection could save numerous short trips to the library, a point particularly important where a library well equipped with the sources of the literature of peptide synthesis is not near at hand. Also, we thought that the envisaged book may be welcome by those who are more versed in English than in German. To the best of our knowledge no similar publication is available.

During their lifetime, especially when growing and dividing, cells go through various steps of the cell cycle. Knowledge of the individual steps of the cell cycle will help us understand the development of a variety of diseases better, including cancer, and also to design new drugs against it. New techniques for studying the molecular basis of these processes have recently been developed and are described in detail in this manual.A glossary helps the reader to cope with the complex cell cycle terminology.

Molecular biological techniques such as DNA/RNA extraction and purification, and especially the polymerase chain reaction, PCR, are rapidly gaining interest also in related fields, such as microbiology or environmental sciences. They offer new approaches and opportunities for the determination of microbial cells, DNA and RNA from soils, roots, rhizospheres, sediments and aquatic environments. Detailed protocols for these applications are described in this manual.

The establishment of microinjection protocols about 20 years ago for cultured cells and shortly thereafter for the generation of transgenic mice by microinjection of DNA into fertilized mouse eggs greatly influ­ enced many fields of biology. Not only have the data generated using these approaches contributed to a large extent to our present under­ standing of gene regulation and cellular function of higher eukaryotic cells, but current knowledge and future developments in this area will certainly have a great impact on basic and applied research for many years to come. This laboratory manual describes the current state of the art in this research area and focuses primarily on both the experimental strategies with an extensive bibliography and the detailed procedures. A large number of studies are presently being performed and a great variety of new experimental designs are rapidly being developed. The book con­ tains protocols on injection of somatic cells as well as on injection of embryos, the use of similar equipment being a common feature. In the articles dedicated to somatic cells, full descriptions of the manual and automatic injection systems are given as well as the methods for the analysis of injected cells by video-microscopy, electron microscopy or in situ hybridizations. In addition, comprehensive protocols are given for injection experiments with very different purposes, such as to study sig­ nal transduction or microtubule dynamics.

Cell culture techniques allow a variety of molecular and cell biological questions to be addressed, offering physiological conditions whilst avoiding the use of laboratory animals. In addition to basic techniques, a wide range of specialised practical protocols covering the following areas are included: cell proliferation and death, in-vitro models for cell differentiation, in-vitro models for toxicology and pharmacology, industrial application of animal cell culture, genetic manipulation and analysis of human and animal cells in culture.

Identification of differentially expressed genes is one of the major challenges in molecular biology. Several techniques allow the cloning of such sequences. However, methods such as RNA subtraction or differential hybridization are time-consuming and require large amounts of mRNA.Recently, a new approach has successfully been developed: Differential-Display Reverse Transcription-PCR (DDRT-PCR). This technique has been proven to be highly effective in identifying sequences that are differentially expressed in various cell types. The most striking advantage is, however, that only nanograms of total RNA are sufficient. Thus every mRNA species expressed in the cell system can be investigated, even those present at very low levels.

New techniques and updated protocols for the detection and analysis of biomolecules - proteins, glycoproteins and nucleic acids - are presented in the second edition of this successful laboratory manual. Highly sensitive systems which are widely used in molecular biological and biomedical laboratories, such as colorimetric, luminescence, fluorescence measuring using antibody/antigen binding or hybridisation, as well as PCR amplification are described in detail. The clearly structured step-by-step protocols with practical hints and a troubleshooting guide are complemented by chapters on the theoretical background and the application of the techniques. Thus the manual will enable scientists to plan, design and conduct the appropriate procedures which fulfill their particular requirements.

Flow cytometric analysis of molecular, biochemical, genetic and developmental parameters using cellular fluorescence techniques as well as fluorescence-activated (FACS) or magnetic (MACS) cell sorting technologies provide unique options for molecular and cellular biology. In recent years, these technologies have been considerably advanced.In this second edition, all chapters have been updated according to the recent improvements and modifications. Further, new protocols have been added, such as on magnetic selection, magnetofluorescent liposomes, the cytometry of secreted products and microbead assays, as well as reporter gene assays for cytometry and cell selection. The recent technical developments allow diagnostic differentiation of cells according to specific gene expression, identification of rare disease-associated cells and isolation of well-defined cells at high purity for cell therapies.

This laboratory manual gives a thorough introduction to basic techniques in Molecular Biology, which can find application in many different fields. It is the result of practical experience, with each protocol having been used extensively in undergraduate courses or tested in the authors laboratory. Thus, they are also likely to work in inexperienced hands the first time they are performed. In addition to detailed protocols and practical notes, each technique includes an overview of its general importance, the time and expense involved in its application and a description of the theoretical mechanisms of each step. This knowledge will enable the users to design their own modifications or to adapt the method to different systems.Surzycki has been teaching undergraduate courses and leading workshops for many years, during which time he has extensively modified and refined the techniques he has described in this manual.

Creating bacteria with modified genetic properties allows the specific investigation of these microorganisms. Electrotransformation is a highly efficient and easy to apply technique to introduce genetic material into bacterial cells. A strong electric field is used for this purpose. In the present manual, protocols for the transformation of about 40 strains of bacteria are described. Emphasis is placed on the individual critical procedural steps, since the practical details mainly depend on the bacterial strain under investigation. This presentation together with the theoretical introductionary chapters, allows the user to modify and adapt each protocol to his/her own experiments. Bacterial strains with relevance in the food industry, biotechnology, medical and veterinary fields, agroindustry and environmental sciences are covered.

Since antibodies tagged with markers have been developed, immunocytochemistry has become the method of choice for identifying tissue substances or for the localisation of nucleic acid in tissue by in situ hybridisation. Resin-embedded tissue is routinely used and new techniques are constantly introduced. Thus, the novice entering these fields has a breathtaking variety of methods open to him. This labmanual covers the embedding of tissue using epoxy resin methods to the more sensitive procedures employing the acrylics. The possibilities and results are discussed so that an understanding of the techniques can be acquired and appropriate choices made. The various resins available and all steps involved in tissue processing, beginning with fixation, as well as the great variety of labelling methods and markers that are commonly used for "on-section" cytochemistry and immunocytochemistry are described, including detailed protocols for the application.

Proteins interacting with diverse ligands - proteins, peptides or DNA - are the basic principles underlying many biological processes, such as antigen-antibody binding, signal transduction or receptor binding.The technique of oligopeptide synthesis on a cellulose membrane and the subsequent binding assays allow the investigation of protein interactions. A particular advantage of these peptide arrays (SPOT - technology) is the high number of oligopeptide probes that can be tested in parallel. Detailed protocols for peptide synthesis, and the analysis of protein-protein, protein-DNA interactions as well as epitope mapping are presented in this manual. It is ideally suited not only for basic research laboratories but also for diagnostic and therapeutic applications since many diseases are related to dysfunctions in protein recognition and binding.

Flow cytometric analysis of molecular, biochemical, genetic and developmental parameters using cellular fluorescence techniques as well as fluorescence-activated (FACS) or magnetic (MACS) cell sorting technologies provide unique options for molecular and cellular biology. In recent years, these technologies have been considerably advanced.In this second edition, all chapters have been updated according to the recent improvements and modifications. Further, new protocols have been added, such as on magnetic selection, magnetofluorescent liposomes, the cytometry of secreted products and microbead assays, as well as reporter gene assays for cytometry and cell selection. The recent technical developments allow diagnostic differentiation of cells according to specific gene expression, identification of rare disease-associated cells and isolation of well-defined cells at high purity for cell therapies.

Creating bacteria with modified genetic properties allows the specific investigation of these microorganisms. Electrotransformation is a highly efficient and easy to apply technique to introduce genetic material into bacterial cells. A strong electric field is used for this purpose. In the present manual, protocols for the transformation of about 40 strains of bacteria are described. Emphasis is placed on the individual critical procedural steps, since the practical details mainly depend on the bacterial strain under investigation. This presentation together with the theoretical introductionary chapters, allows the user to modify and adapt each protocol to his/her own experiments. Bacterial strains with relevance in the food industry, biotechnology, medical and veterinary fields, agroindustry and environmental sciences are covered.

Since antibodies tagged with markers have been developed, immunocytochemistry has become the method of choice for identifying tissue substances or for the localisation of nucleic acid in tissue by in situ hybridisation. Resin-embedded tissue is routinely used and new techniques are constantly introduced. Thus, the novice entering these fields has a breathtaking variety of methods open to him. This labmanual covers the embedding of tissue using epoxy resin methods to the more sensitive procedures employing the acrylics. The possibilities and results are discussed so that an understanding of the techniques can be acquired and appropriate choices made. The various resins available and all steps involved in tissue processing, beginning with fixation, as well as the great variety of labelling methods and markers that are commonly used for "on-section" cytochemistry and immunocytochemistry are described, including detailed protocols for the application.

Designed as an introductory text the authors cover all core strategies in the application of modern recombinant DNA technology. The first chapters directly address the applications of polymerase chain reaction to a variety of problems in DNA cloning that are, or have been, extremely challenging using more traditional approaches and technologies. These include cDNA cloning and transcript mapping, mutagenesis as well as the cloning of very long transcripts and protocols using limiting amounts of total RNA. Further chapters describe approaches to subtractive cloning technologies as well as novel specialized expression cloning and library screening strategies. The handbook contains detailed step-by-step protocols and extensive hands-on advice.

The study of signal transduction mechanisms has become one of the most important branches of biomedical science, indispensable to understanding the normal actions of hormones, neurotransmitters and other extracellular signaling molecules as well as many pathophysiological processes, including inflammation and cancer. In this volume, the various techniques for measuring Ca2+ in the cytosol, in the various organelles, and in the immediate surroundings of individual cells are described in practical detail by experts. Also included is a chapter on tracking calmodulin inside cells using fluorescence technology. A Springer Lab Manual

Fluorescence in situ hybridization (FISH) has been developed as a powerful technology which allows direct visualisation or localisation of genomic alterations. The technique has been adopted to a range of applications in both medicine, especially in the areas of diagnostic cytogenetics, and biology. Topics described in this manual include: FISH on native human tissues, such as blood, bone marrow, epithelial cells, hair root cells, amniotic fluid cells, human sperm cells; FISH on archival human tissues, such as formalin fixed and paraffin embedded tissue sections, cryofixed tissue; simultaneous detection of apoptosis and xpression of apoptosis-related genes; comparative genomic ybridization; and special FISH techniques.

Electrophoresis is a powerful method to analyze nucleic acids (DNA, RNA). Various sophisticated techniques such as capillary electrophoresis, pulsed-field electrophoresis, fingerprinting using RFLP and RAPD, DNA sequencing, and mobility shift assay are described in detail. The required apparatus, appropriate use, preparation of probes, gel staining, interpretation of results, tricks for troubleshooting, manufacturers addresses, helpful Internet resources as well as specific applications, e.g. in legal medicine, microbiology and agriculture, are presented by leading experts.

"Protein Structure Analysis - Preparation and Characterization" is a compilation of practical approaches to the structural analysis of proteins and peptides. Here, about 20 authors describe and comment on techniques for sensitive protein purification and analysis. These methods are used worldwide in biochemical and biotechnical research currently being carried out in pharmaceu­ tical and biomedical laboratories or protein sequencing facilities. The chapters have been written by scientists with extensive ex­ perience in these fields, and the practical parts are well documen­ ted so that the reader should be able to easily reproduce the described techniques. The methods compiled in this book were demonstrated in student courses and in the EMBO Practical Course on "Microsequence Analysis of Proteins" held in Berlin September 10-15, 1995. The topics also derived from a FEBS Workshop, held in Halkidiki, Thessaloniki, Greece, in April, 1995. Most of the authors participated in these courses as lecturers and tutors and made these courses extremely lively and successful. Since polypeptides greatly vary depending on their specific structure and function, strategies for their structural analysis must for the most part be adapted to each individual protein. Therefore, advantages and limitations of the experimen­ tal approaches are discussed here critically, so that the reader becomes familiar with problems that might be encountered.

Researchers may find themselves confronted with proteases, either because they play an essential role in a particular process they are studying, or because they interfere with that process. In either case they may need to investigate or inhibit the proteolytic activity. Others may wish to use proteolytic enzymes as laboratory tools.This book has been written with these investigators in mind and includes assay methods using natural and artificial substrates, genetic-based assays, and strategies for the inhibition, purification and crystallization of proteases. In selected chapters the use of proteolytic enzymes to analyze proteins, segregate cells or in peptide synthesis is covered.

Standard molecular biology techniques are used widely in biomedical re­ search and have had a major impact on the understanding of physiologic processes and disease mechanisms. They also play an increasing role in mo­ lecular genetic diagnosis and new therapeutic approaches. This laboratory manual is designed for researchers who need reliable pro­ tocols for their laboratorywork. It provides a step by step approach to stan­ dard methods of molecular biology. A wide range of topics is covered by protocols for immediate application in the laboratory. These include pur­ ification, isolation, labeling, hybridization, sequencing and enzymatic mod­ ification ofDNA or RNA, polymerase chain reaction, cloning, libraries, and protein techniques. The manual is based on experience with a practical course in molecular biologytechniques, which was taught bythe authors. Since most methods in molecular biology recapitulate in a test tube mechanisms that occur natu­ rally in the cell, the first three chapters describe and illustrate basic mechan­ isms of molecular biology. U nderstanding more about these basic mechan­ isms will enable the researcher to apply techniques more specifically, based on the knowledge of their potential and limits. W e hope that users find this manual a helpful guide on their road to exciting insights.