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This volume expands upon the previous edition with current, detailed protocols for investigating membranes and their component lipids in artificial membranes, cells, and in silico. Chapters focus on properties of the component lipids, membranes and their biophysical properties, fluorescent probes for studying membranes, sample preparation, physical techniques to study membrane composition, properties , and function, behavior of cholesterol within a bilayer and examination of cholesterol-dependent phase separation. 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 practical, Methods in Membrane Lipids, Second Edition seeks to aid scientist in further study into membrane lipids.
The first edition of Protein Purification Protocols (1996), edited by Professor Shawn Doonan, rapidly became very successful. Professor Doonan achieved his aims of p- ducing a list of protocols that were invaluable to newcomers in protein purification and of significant benefit to established practitioners. Each chapter was written by an ex- rienced expert in the field. In the intervening time, a number of advances have w- ranted a second edition. However, in attempting to encompass the recent developments in several areas, the intention has been to expand on the original format, retaining the concepts that made the initial edition so successful. This is reflected in the structure of this second edition. I am indebted to Professor Doonan for his involvement in this new edition and the continuity that this brings. Each chapter that appeared in the original volume has been reviewed and updated to reflect advances and bring the topic into the 21st century. In many cases, this reflects new applications or new matrices available from vendors. Many of these have increased the performance and/or scope of the given method. Several new chapters have been introduced, including chapters on all the currently used protein fractionation and ch- matographic techniques. They introduce the theory and background for each method, providing lists of the equipment and reagents required for their successful execution, as well as a detailed description of how each is performed.
The principle that antibodies can be used as cytochemical agents provided they are tagged with suitable markers has been evident for over 50 years. During this time the use of immunocytochemical meth ods has spread to a wide array of biological disciplines. Early applica tions focused on the detection of microbial antigens in tissues, while more recent applications have used monoclonal antibodies to study cell differentiation during embryonic development. For a select few disci plines, volumes have been published focusing on the specific applica tion of immunocytochemical techniques to that discipline. What distinguishes the present book, Immunocytochemical Meth ods and Protocols, from earlier books is its broad appeal to researchers in all disciplines, including those in both research and clinical settings. The methods and protocols presented here are designed to be general in their application and the accompanying "Notes" provide invaluable assistance in adapting or troubleshooting the protocols. Interspersed throughout the book are chapters providing overviews of select topics related to immunocytochemistry.
The ability to introduce macromolecules into animal cells, includ ing DNA, RNA, proteins, and other bioactive compounds has facili tated a broad range of biological studies, from biochemistry and biophysics to molecular biology, cell biology, and whole animal stud ies. Gene transfer technology in particular will continue to play an essential role in studies aimed at improving our understanding of the relationships between the gene structure and function, and it has impor tant practical applications in both biotechnology and biomedicine, as evidenced by the current intense interest in gene therapy. Although DNA and other macromolecules may be introduced into cells by a variety of methods, including chemical treatments and microinjection, el- troporation has proven to be simpler to perform, more efficient, and effective with a wider variety of cell types than other techniques. The early and broad success of electric field-mediated DNA transfer soon prompted researchers to investigate electroporation for transferring other types of molecules into cells, including RNA, enzymes, antibodies, and analytic dyes. Animal Cell Electroporation and Electrofusion Protocols begins with three chapters that describe the theoretical and practical aspects of electroporation, including a review of the commercially available instrumentation. These introductory chapters will be of particular inter est to those new to electric field technologies and to those developing protocols for as yet untested species or cell types. Nineteen chapters follow that present well-tested protocols for electroporation of proteins and DNA into insect, fish, and mammalian cells.
This comprehensive collection of recently developed methods for producing new antibody reagents by immunization and recombinant DNA techniques contains ready-to-use protocols that illuminate current areas of research on antibody structure, functions, and applications. The methods can be applied in basic immunological studies involving antibody specificity, catalysis, and evolution, and in the isolation of rare antibodies by phage display technology and the engineering of new antibodies by mutagenesis. They offer insight into new ways of developing clinically useful antibody reagents. Antibody Engineering Protocols constitutes a single-source volume for laboratory investigators who want to minimize extensive literature and methodology searches and to work productively in their fields with reproducible step-by-step protocols.
When first conceived, not only was the aim of Protocols for Oligo nucleotides and Analogs to provide wide coverage of the ohgonuc- otide chemistry field for readers who are well versed within the field, but also to give investigators just entering into the field a new perspective. The very first book on this topic was edited and published by Michael Gait in 1984, in whose laboratory I encountered the newer aspects of oligonucleotide chemistry. Since then, oligonucleotide research has developed to such an extent that its uses extend far beyond basic studies, and now find wide application throughout clinical science as well. Until recently, the major application of oligonucleotides has been in the area of DNA-based diagnostic and "antisense oligonucleotid- based therapeutic approaches. However, oligonucleotides are now also being used as therapeutic agents and are thus frequently found in clinical trials in humans. Synthesis of unmodified oligonucleotides using automated synthe sizers has become a common practice in numerous laboratories. How ever, improvements on the existing techniques and the introduction of ever newer methods for oligonucleotide synthesis is constantly driving ahead in the leading research laboratories. And several new oligonucle otide analogs have been synthesized and studied for their individual prop erties in recent years. The present volume strives to bring the readers the most up-to-date information on the newest aspects of synthesis of oligo nucleotides and their analogs. A separate volume covers synthesis of oligonucleotide conjugates, along with most of the analytical techniques presently used for analysis of oligonucleotides.
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.
The purpose of DNA Sequencing Protocols is to provide detailed practical procedures for the widest range of DNA sequencing meth ods, and we believe that all the vanguard techniques now being applied in this fast-evolving field are comprehensively covered. Sequencing technology has advanced at a phenomenal rate since the original methods were first described in the late 1970s and there is now a huge variety of strategies and methods that can be employed to determine the sequence of any DNA of interest. More recently, a large number of new and innovative sequencing techniques have been developed, including the use of such novel polymerases as Tag poly merase and Sequenase, the harnessing of PCR technology for linear amplification (cycle) sequencing, and the advent of automated DNA sequencers. DNA sequencing is surely one of the most important techniques in the molecular biology laboratory. Sequence analysis is providing an increasingly useful approach to the characterization of biological systems, and major multinational projects are already underway to map and sequence the entire genome of organisms, such as Escherichia coli, Saccharomyces cerevisiae, Caenorhabditis elegans, and Homo sapiens. Most scientists recognize the importance of DNA sequence data and perceive DNA sequencing as a valuable and indispensable aspect of their work. Recent technological advances, especially in the area of automated sequencing, have removed much of the drudg ery that was formerly associated with the technique, whereas innova tive computer software has greatly simplified the analysis and manipulation of sequence data.
Chromosomes, as the genetic vehicles, provide the basic material for a large proportion of genetic investigations, from the construction of gene maps and models of chromosome organization, to the inves tigation of gene function and dysfunction. The study of chromosomes has developed in parallel with other aspects of molecular genetics, beginning with the first preparations of chromosomes from animal cells, through the development of banding techniques, which permitted the unequivocal identification of each chromosome in a karyotype, to the present analytical methods of molecular cytogenetics. Although some of these techniques have been in use for many years, and can be learned relatively easily, most published scientific reports—as a result of pressure on space from editors, and the response to that pressure by authors—contain little in the way of technical detail, and thus are rarely adequate for a researcher hoping to find all the necessary information to embark on a method from scratch. A new user needs not only a detailed description of the methods, but also some help with problem solving and sorting out the difficulties en countered in handling any biological system. This was the require ment to which the series Methods in Molecular Biology is addressed, and Chromosome Analysis Protocols forms a part of this series.