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In Vitro Mutagenesis Protocols
Hands-on researchers with proven track records describe in stepwise fashion their advanced mutagenesis techniques. The contributors focus on improvements to conventional site-directed mutagenesis, including a chapter on chemical site-directed mutagenesis, PCR-based mutagenesis and the modifications that allow high throughput mutagenesis experiments, and mutagenesis based on gene disruption (both in vitro- and in situ-based). Additional methods are provided for in vitro gene evolution; for gene disruption based on recombination, transposon, and casette mutagenesis; and for facilitating the introduction of multiple mutations. Time-tested and highly practical, the protocols in In Vitro Mutagenesis Protocols, 2nd Edition offer today's molecular biologists reliable and powerful techniques with which to illuminate the proteome.
Two-Hybrid Systems
The yeast two-hybrid system is one of the most widely used and productive techniques available for investigating the macromolecular interactions that affect virtually all biological processes. In Two-Hybrid Systems: Methods and Protocols, Paul N. MacDonald has assembled a collection of these powerful molecular tools for examining and characterizing protein-protein, protein-DNA, and protein-RNA interactions. The techniques range from the most basic (introducing plasmids into yeasts, interaction assays, and recovering the plasmids from yeast) to the most advanced alternative strategies (involving one-hybrid, split two-hybrid, three-hybrid, membrane recruitment systems, and mammalian systems). ...
PCR Mutation Detection Protocols
1Bimal D. Theophilus and Ralph Rapley provide biological and clinical investigators with a comprehensive collection of new, recent, and updated PCR-based screening methods suitable for detecting the presence of both known and novel mutations. The methods cover point mutations (e.g., ASO-PCR, SSCP, DGGE, chemical cleavage), deletions (multiplex PCR, FISH, blotting), non-sense mutations (PTT), and more. The new and exciting techniques of DNA array analysis, along with such recently developed experimental methods as conformation-sensitive gel electrophoresis, are also included. Each chapter explains the basic theory behind the technique and provides valuable notes essential for its successful execution.
DNA Methylation Protocols
DNA Methylation Protocols offer a set of readily reproducible protocols of the analysis of DNA methylation and methylases. These powerful methods provide the tools necessary for studying methylation at both the global level and the level of sequence, and include many techniques for identifying genes that might be aberrantly methylated in cancer and aging. Additional methods cover genome-wide analysis of abnormal DNA methylation and the isolation and measurement of demethylases and related proteins.
Calcium-Binding Protein Protocols
Calcium plays an important role in a wide variety of biological processes. This divalent metal ion can bind to a large number of proteins; by doing so it modifies their biological activity or their stability. Because of its distinct che- cal properties calcium is uniquely suited to act as an on–off switch or as a light dimmer of biological activities. The two books entitled Calcium-Binding Protein Protocols (Volumes I and II) focus on modern experimental analyses and methodologies for the study of calcium-binding proteins. Both extracel- lar and intracellular calcium-binding proteins are discussed in detail. H- ever, proteins involved in calcium handling (e. g. , calcium pumps and calcium ...
Oxidative Stress Biomarkers and Antioxidant Protocols
The first protocols book, Free Radical and Antioxidant Protocols (1) was published in late 1998. Sections were divided into three parts, covering selected biochemical techniques for measuring oxidative stress, antioxidant (AOX) activity, and combined applications. In choosing the 40 methods to be included in that book, I realized there were considerably more of equal value than that which we could have presented in a single volume. To produce a comprehensive resource, this book and a third are being compiled to expand coverage of the field. A summary of papers (2) published on this important subject emphasizes the continuing rapid growth in oxidative stress investigations relating to our understanding of biochemical reactions, their relevance to pathophysiological mechanisms, how disease may arise, and how therapeutic intervention may be achieved(3). Although there is some overlap between the categories, the ana- sis shown below illustrates where current studies are concentrated and are almost evenly distributed between free radicals and AOX. Over the last 4 yr, there has been a 55% increase in the number of papers published in the area.
Epithelial Cell Culture Protocols
There have been significant advances in research involving the isolation and culture of epithelial cells in the past decade, and many new techniques have been developed. Monolayer cultures can be used to evaluate the nature and behavior of cells, while the use of epithelial cells in model systems has allowed a deeper understanding of cellular and molecular mechanisms and interactions. The aim of this book is to provide a comprehensive, step-by-step guide to many techniques for epithelial cell culture, combining in one volume the more commonly used protocols along with many that are more speci- ized. Epithelial Cell Culture Protocols should help those who are new to this field and want to lea...
In Situ Detection of DNA Damage
Detection and analysis of DNA damage is of critical importance in a variety of biological disciplines studying apoptosis, cell cycle and cell di- sion, carcinogenesis, tumor growth, embryogenesis and aging, neu- degenerative and heart diseases, anticancer drug development, environmental and radiobiological research, and others. Individual cells within the same tissue or in cell culture may vary in the extent of their DNA damage and, consequently, can display different re- tions to it. These differences between individual cells in the same cell popu- tion are detected using in situ approaches. In situ is a Latin term meaning “on site” or “in place.” It is used to denote the processes ...
Embryonic Stem Cells
It is fair to say that embryonic stem (ES) cells have taken their place beside the human genome project as one of the most discussed biomedical issues of the day. It also seems certain that as this millennium unfolds we will see an increase in scientific and ethical debate about their potential utility in society. On the scientific front, it is clear that work on ES cells has already generated new possibilities and stimulated development of new strategies for increasing our und- standing of cell lineages and differentiation. It is not naïve to think that, within a decade or so, our overall understanding of stem cell biology will be as revolutionized as it was when the pioneering hemopoietic...
Neural Stem Cells
Over the last decade, neural stem cell research has provided penetrating insights into the plasticity and regenerative potential of the brain. Stem cells have been isolated from embryonic as well as adult central nervous system (CNS). Many non-CNS mammalian tissues also contain stem cells with a more limited repertoire: the replacement of tissue-specific cells throughout the li- time of the organism. Progress has been made in understanding fundamental stem cell properties that depend on the interplay of extrinsic signaling factors with intrinsic genetic programs within critical time frames. With this growing knowledge, scientists have been able to change a neural stem cell’s fate. - der ce...
