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In this completely updated and expanded edition of a classic bench manual, hands-on experts take advantage of the latest advances in ribozyme, DNAzyme, hammerhead ribozymes and derivatives, and RNA interference technologies to describe in detail the exciting and successful methods now available for gene inactivation in vitro and in vivo. Their optimized techniques employ hairpin ribozymes, DNAzymes, hammerhead ribozymes and derivatives, group I intron ribozymes, RNase P ribozymes, and siRNAs, as well as general methods for RNA structure analysis, delivery of oligonucleotides, and gene therapy. Also provided are novel methods for identifying accessible cellular mRNA sites; group I intron and RNAse P ribozyme protocols for effective design, selection, and therapeutic applications; and the latest RNAi methods for sequence-specific gene silencing in a wide variety of organisms. Additional techniques cover the analysis of ribozyme structures and conformational transitions using nucleotide analog interference mapping and fluorescence resonance energy transfer, the use of ribozymes in clinical and gene therapy, and the use of ribozymes and DNAzymes in rodent models of human disease. Each proven protocol includes a background introduction outlining the principle behind the technique, step-by-step instructions, lists of equipment and reagents, and tips on troubleshooting and avoiding known pitfalls. Comprehensive and up-to-date, Ribozymes and siRNA Protocols details for experienced and novice investigators alike the many exciting advances in our understanding of nucleic acid enzymes, as well as demonstrating how they may be used to analyze gene function and target validation, and to productively develop novel therapeutics for human diseases.

Cytology. --- Cell Biology. --- Cell biology --- Cellular biology --- Biology --- Cells --- Cytologists

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The discovery that trinucleotide repeats significantly influence the age of onset and severity of a variety of hereditary neurological disorders has opened the door to a deeper understanding of the disease mechanisms involved, as well as to a more productive search for novel therapeutic interventions. In Trinucleotide Repeat Protocols, established leaders in trinucleotide repeat disease describe in step-by-step detail their best techniques for studying trinucleotide pathology at the molecular level. The protocols cover a variety of targets, ranging from DNA and RNA to proteins and whole animals, and focus not only on causal genes, but also on their consequent products, such as transcription factors, neurotransmitter receptors, proteasomes, and mitochondria/oxidation damage. Experimental systems employed include E. coli, yeast, C. elegans, mouse, and generally take a clinical point of view. The authors utilize a wide range of techniques, including gel electrophoresis, quantitative RT-PCR, immunological analysis, antibody usage and its applications, receptor assays using radioisotope handling, gene delivery by virus, brain cell and organotypic cultures, gender dependency, and neuron structure analysis. Each protocol follows the successful Methods in Molecular Biology™ series format, offering step-by-step laboratory instructions, an introduction outlining the principle behind the technique, lists of the necessary equipment and reagents, and tips on troubleshooting and avoiding known pitfalls. Cutting-edge and highly practical, Trinucleotide Repeat Protocols offers neuroscientists powerful tools to elucidate both normal brain function and the mechanisms of hereditary neurological disease, as well as to develop the next generation of therapies for neuronal genetic diseases.

Cytology. --- Cell Biology. --- Cell biology --- Cellular biology --- Biology --- Cells --- Cytologists

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Germ cell research has produced enormous advances in recent years and more recently has entered into an explosive phase of new discoveries with the introduction of transgenic technologies and nuclear cloning. In Germ Cell Protocols, expert laboratory investigators describe in step-by-step detail a series of powerful techniques for the molecular and genetic analysis of germ cells in a variety of different reproductive systems. Chosen for their proven success in advancing our understanding of germ cells, these methods cover sperm and egg activation, motility, fertilization, nuclear development, nuclear cloning, the molecular characterization of specific events, and the imaging of cell structures. Volume 1: Sperm and Oocyte Analysis focuses on sperm cells, oocyte analysis, oocyte maturation, fertilization, and preparation techniques. Highlights include in vitro maturation and fertilization of human, porcine, and canine oocytes; the cryopreservation of sperm cells; establishment of an in vitro spermatogenesis system; visualization of sperm accessory structures; and motility assays of spermatozoa. Each readily reproducible protocol is described in step-by-step detail and contains an introduction outlining the principle behind the technique, lists of equipment and reagents, and tips on troubleshooting and avoiding known pitfalls. An accompanying second volume, Molecular Embryo Analysis, Live Imaging, Transgenesis, and Cloning, contains methods for molecular egg analysis, live egg imaging, nuclear cloning, oocyte preservation, and nuclear transfer. Comprehensive and cutting-edge, Germ Cell Protocols offers both novice and established researchers a gold-standard collection of hard-to-find methods for high impact research, diverse procedures that are easy-to-follow, well-illustrated, and allow a cross-species transfer of knowledge from lower vertebrates to higher mammalian systems.

Cytology. --- Cell Biology. --- Cell biology --- Cellular biology --- Biology --- Cells --- Cytologists

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Contemporary approaches to the synthesis of chemically modified proteins not only require efficient means to control conjugation and the specific site of attachment of the conjugated moiety, but also the effective use of recent developments in microarray technologies and nanobiotechnology. In Bioconjugation Protocols: Strategies and Methods, expert laboratorians update the classic methods and introduce valuable new approaches that go beyond basic conjugation techniques to include elements from advanced organic synthesis, molecular biology, and materials science. These readily reproducible methods cover the preparation of protein conjugates using covalent and noncovalent conjugation, the synthesis of nucleic acid conjugates using a variety of labeling techniques, and approaches to semisynthetic conjugates of proteins. Additional chapters address the biofunctionalization of inorganic surfaces, including the on-chip synthesis of peptide nucleic acids to generate microarrays for the high-throughput analysis of RNA and DNA, gold nanoparticles, and carbon nanotube probes for atomic force microscopy. All the protocols follow the successful Methods in Molecular Biology™ series format, each one offering step-by-step laboratory instructions, an introduction outlining the principle behind the technique, lists of the necessary equipment and reagents, and tips on troubleshooting and avoiding known pitfalls. Cutting-edge and highly practical, Bioconjugation Protocols: Strategies and Methods offers both novice and experienced researchers access to the broad array of techniques needed to master the semisynthesis of functional biomolecular reagents for a wide variety of applications-ranging from novel biomedical diagnostics, to powerful new therapeutics, to advanced biomaterials.

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In this second edition of a widely appreciated work, Receptor Signal Transduction Protocols, a panel of internationally recognized investigators presents their best methods for studying G-protein-coupled receptors (GPCRs) and events immediately downstream of their activation. This new edition combines updates of key chapters from the first edition with a large number of new contributions on the many successful methodologies that have emerged more recently. The methods are focused primarily on events at the receptor level, including ligand binding, on the genetic manipulation of receptors, the generation of model cell lines in which to study them, and the interaction and activation of G-proteins. Additional methods concentrate on receptor expression and localization, receptor internalization and post-translational modification, GPCR-protein interactions, and the use of knock-out and knock-in strategies for determining the physiological roles of receptors. The laboratory protocols follow the successful Methods in Molecular Biology™ series format, each one offering step-by-step laboratory instructions, an introduction outlining the principle behind the technique, lists of equipment and reagents, and tips on troubleshooting and avoiding known pitfalls. Comprehensive and highly practical, Receptor Signal Transduction Protocols, Second Edition offers both novice and experienced investigators powerful cutting-edge techniques that provide an array of approaches and specific methods that will aid in the understanding of GPCR structure and function.

Cytology. --- Cell Biology. --- Cell biology --- Cellular biology --- Biology --- Cells --- Cytologists