Macromolecular Crystallography Protocols, now in two volumes, examines major developments that have occurred since publication of the acclaimed first edition nearly a decade ago. Volume 1 is composed of detailed protocols for the preparation and optimization of crystals. Volume 2 complements the first volume by addressing laboratory techniques for crystal handling and structural characterization. The volume concludes with a survey of available crystallographic software.

In the decade since publication of the first edition this book, the field has seen several major developments. These developments have both accelerated the pace of structure determination and made crystallography accessible to a broader range of investigators. Volume I is dedicated to crystallization and ways to increase the odds of obtaining crystals in macromolecules. Volume 2 covers both computational methods for characterizing crystals and solving structures.

Protein engineering is a fascinating mixture of molecular biology, protein structure analysis, computation, and biochemistry, with the goal of developing useful or valuable proteins. Protein Engineering Protocols will consider the two general, but not mutually exclusive, strategies for protein engineering. The first is known as rational design, in which the scientist uses detailed knowledge of the structure and function of the protein to make desired changes. The s- ond strategy is known as directed evolution. In this case, random mutagenesis is applied to a protein, and selection or screening is used to pick out variants that have the desired qualities. By several rounds of mutation and selection, this method mimics natural evolution. An additional technique known as DNA shuffling mixes and matches pieces of successful variants to produce better results. This process mimics recombination that occurs naturally during sexual reproduction. The first section of Protein Engineering Protocols describes rational p- tein design strategies, including computational methods, the use of non-natural amino acids to expand the biological alphabet, as well as impressive examples for the generation of proteins with novel characteristics. Although procedures for the introduction of mutations have become routine, predicting and und- standing the effects of these mutations can be very challenging and requires profound knowledge of the system as well as protein structures in general.

Protein Phosphatase Protocols presents a broad range of protocols for the study of protein phosphatases, all written by experts and innovators from phosphatase laboratories around the world. This volume is a compendium of resources for the study of protein phosphatases and their potential as drug targets. Experimental methodologies are taken from proteomics, bioinformatics, genomics, biochemistry, RNAi, and genetics.

Covering experiment and theory, bioinformatics approaches, and state-of-the-art simulation protocols for better sampling of the conformational space, this volume describes a broad range of techniques to study, predict, and analyze the protein folding process. Protein Folding Protocols also provides sample approaches toward the prediction of protein structure starting from the amino acid sequence, in the absence of overall homologous sequences.

Agrobacterium tumefaciens is a soil bacterium that for more than a century has been known as a pathogen causing the plant crown gall disease. Unlike many other pathogens, Agrobacterium has the ability to deliver DNA to plant cells and permanently alter the plant genome. The discovery of this unique feature 30 years ago has provided plant scientists with a powerful tool to genetically transform plants for both basic research purposes and for agricultural development. Compared to physical transformation methods such as particle bomba- ment or electroporation, Agrobacterium-mediated DNA delivery has a number of advantages. One of the features is its propensity to generate a single or a low copy number of integrated transgenes with defined ends. Integration of a single transgene copy into the plant genome is less likely to trigger “gene silencing” often associated with multiple gene insertions. When the first edition of Agrobacterium Protocols was published in 1995, only a handful of plants could be routinely transformed using Agrobacterium. Agrobacterium-mediated transformation is now commonly used to introduce DNA into many plant species, including monocotyledon crop species that were previously considered non-hosts for Agrobacterium. Most remarkable are recent developments indicating that Agrobacterium can also be used to deliver DNA to non-plant species including bacteria, fungi, and even mammalian cells.

These volumes review the most current methods for drug target discovery and validation. They explore how recent improvement in understanding the molecular mechanisms of human pathology is impacting drug target discovery in the laboratory and in real therapeutics, specifically for cancers and autoimmune disorders. This book provides a thorough review of the most cutting-edge methods available for each step in drug target identification, validation, and clinical application.

Glycobiology involves studies of complex carbohydrates and posttrans- tional modifications of proteins, and has become an important interdiscip- nary field encompassing chemistry, biochemistry, biology, physiology, and pathology. Although initial research was directed toward elucidation of the different carbohydrate structures and the enzymes synthesizing them, the field has now moved toward identifying the functions of carbohydrates. The pro- cols described in Glycobiology Protocols form a solid basis for investigations of glycan functions in health and disease. The cloning of many of the genes participating in glycosylation processes has helped to enhance our knowledge of how glycosylation is controlled, but has also added another dimension of complexity to the great heterogeneous variety of the structures of the oligos- charides of glycoproteins, proteoglycans, and glycolipids. A family of similar enzyme proteins exists for each glycosylation step. Glycosyltransferases are extremely specific for both the nucleotide sugar donor and the acceptor s- strate, but many other factors control sugar transfer, including the locali- tion and topology of enzymes, cofactors, possible chaperone proteins, and the availability of sugar acceptor substrates. The analysis of the intracellular organization of glycosylation and of the factors controlling the activities of the participating enzymes in the cell are important areas that need more research efforts. Another challenge for future research is to understand the glycodynamics of a cell, that is, how the cell responds to stimuli leading to biological and pathological changes in terms of alterations in glycosylation, and how this affects the biology of the cell.

Nuclear Transfer Protocols: Cell Reprogramming and Transgenesis is a comprehensive review of nuclear transfer technology in vertebrates, aimed at reprogramming differentiated nuclei and effecting targeted gene transfer. The emphasis here is on providing readily reproducible techniques for the gene- tion of cloned embryos and animals in a number of key research and commercially important vertebrates. Additional chapters provide alternative cutting-edge methods for nuclear transfer, such as zona-free nuclear transfer and serial nuclear transfer. Of immense practical benefit are descriptions of procedures associated with cloning, such as in vitro maturation of oocytes, activation and culture of cloned embryos, maintenance of pregnancy, and neonatal care of clones. Nuclear Transfer Protocols: Cell Reprogramming and Transgenesis also provides an understanding of the factors involved in nuclear reprogramming, which is imperative for the success of cloning. A section dealing with such cloning-related issues as aging and normality of clones is also included making this an essential comprehensive handbook for research and commercial labo- tories involved in, or intending to work on, nuclear transfer. The volume will prove beneficial to molecular biologists, stem cell biologists, clinicians, biotechnologists, students, veterinarians, and animal care technicians involved with reprogramming, nuclear transfer, and transgenesis.

This book presents detailed protocols for the multidisciplinary application of Pyrosequencing® technology, all written by world-renowned experts. This comprehensive volume enables quick reference by collecting the primary applications for Pyrosequencing®, and supplementing each protocol with troubleshooting tips specific to that method. This volume both highlights the versatility of and provides detailed protocols for the application of Pyrosequencing®.