In Protein Lipidation Protocols, Michael Gelb brings together a collection of readily reproducible techniques for studying protein lipidation, the covalent attachment of lipids to proteins. These cutting-edge methods-many never published before in a "hands-on" format-deal with glycosyl phosphatidylinositol (GPI)-containing compounds, protein fatty acylation, and protein prenylation. Included are novel techniques for determining the chemical structure of GPI-anchors, for radiolabeling the prenyl groups of protein in eukaryotic cells, a tool for developing inhibitors of the protein farnesyltransferase, and for an exciting lysosomal enzyme that cleaves fatty acyl groups from proteins, the first fatty acylase discovered. Protein Lipidation Protocols offers biochemists, cell and molecular biologists, medicinal chemists, and pharmaceutical researchers state-of-the-art tools for understanding the complex biochemistry of protein lipidation, as well as catalyzing the development of many important new biopharmaceuticals, including anticancer drugs.

This volume explores techniques used to detect lipids attached to proteins, to analyze the function of lipid modifications, and to characterize the enzymes that add and remove lipids from proteins. The book is organized into seven parts: Part One describes chemically-based strategies to identify substrates for protein lipidation that can be applied to individual proteins or globally using proteomics. Part Two focuses on the enzymes that remove fatty acids from proteins and provides methods to monitor protein biogenesis and palmitate turnover. Part Three addresses biochemical and cellular characterization of DHHC S-acyltransferases, a family of enzymes with 23 members encoded by the human genome. Part Four presents the SwissPalm 2 database and tips on how to use it effectively. Part Five focuses on fatty acylation that occurs in the lumen of the secretory pathway. Parts Six and Seven conclude the book with methods to produce and assay lipid-modified and integral membrane proteins. 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. Cutting-edge and authoritative, Protein Lipidation: Methods and Protocols is a valuable resource for experts in the field and for investigators who encounter protein lipidation through their research on a particular cellular process or favorite protein.

Concise chapters, written by experts in the field, cover a wide spectrum of topics on lipid and membrane formation in microbes (Archaea, Bacteria, eukaryotic microbes).All cells are delimited by a lipid membrane, which provides a crucial boundary in any known form of life. Readers will discover significant chapters on microbial lipid-carrying biomolecules and lipid/membrane-associated structures and processes.

Adhesion molecules are of fundamental importance in the regulation of immunity, inflammation, tissue remodeling, and embryonic development. They comprise different families of homologous proteins, such as selectins, integrins, cadherins, and immunoglobins. In addition, beyond these groups, other str- tures with adhesive properties, such as proteoglycans, occludin, and CD44, have been characterized recently. An understanding of the type and characteristics of adhesive molecules expressed by the different cell types and the possibility of manipulating their activity promises considerable clinical potential. Antibodies, small peptidic and nonpeptidic molecules, have recently been used to inhibit thrombosis by blocking platelet aggregation or inflammation through inhibition of leukocyte infiltration and adhesion. Inhibitors of adhesive molecules are used in expe- mental systems for the study of tumor growth and dissemination. Among major goals in the field are the identification of new members of the known adhesive protein families and of independent new adhesive structures. After structural characterization, even more demanding is the study of the biological activity of the new proteins, and the development of simple, rapid tests for the screening of possible inhibitors. In this regard, the production of such reagents as fragments and antibodies would help define the structure–function relati- ship of individual proteins. Data available in the literature show the complexity of the adhesive process and how different molecular epitopes might contribute to the adhesive properties of a single structure. Finally, a new area of investi- tion is the characterization of the intracellular signaling cascade triggered by the engagement of transmembrane adhesive proteins.

As key components of many cell signaling pathways, protein kinases are implicated in a broad variety of diseases, including cancers and neurodegenerative conditions, and offer considerable potential as tractable targets for therapeutic intervention. In Protein Kinase Protocols, a panel of highly skilled laboratory investigators describe both basic and more sophisticated methods for the analysis of kinase-mediated signaling cascades, with emphasis on the identification of proteins according to their interactive relationships and the analysis of their functional properties. Described in step-by-step detail, these readily reproducible techniques offer novices quick access to a complicated field, and provide more experienced investigators many novel time-saving ploys. Emphasis is given to the critical technical steps that are often omitted from methods published in the primary literature. There are also tips on potential pitfalls and copious notes on how to adjust the protocols to work in related systems. Broad in its range of techniques and thoroughly detailed to help ensure experimental success, Protein Kinase Protocols offers both novice and experienced investigators powerful tools for understanding the functional roles of specific protein kinases within signaling cascades and for identification and evaluation of novel therapeutic targets.

The molecular characterization of RNA and its interactions with proteins is an important and exciting area of current research. Organisms utilize a variety of RNA–protein interactions to regulate the expression of their genes. This is particularly true for eukaryotes, since newly synthesized messenger RNA must be extensively modified and transported to the cytoplasm before it can be used for protein synthesis. The realization that posttranscriptional processes are critical components of gene regulation has sparked an explosion of interest in both stable ribonucleoprotein (RNP) complexes and transient RNA–protein interactions. RNA is conformationally flexible and can adopt complex structures that provide diverse surfaces for interactions with proteins. The fact that short RNA molecules (aptamers; see Chapter 16) can be selected to bind many different types of molecules is evidence of the structural variability of RNA. RNA molecules are rarely entirely single- or double-stranded, but usually contain multiple short duplexes interrupted by single-stranded loops and bulges; in some RNAs, such as tRNAs, the short duplexes stack on each other. Further variability is generated by the presence of non-Watson-Crick base pairs, modified nucleotides, and more complex structures, such as pseudoknots and triple-strand interactions.

Lipid Modification of Proteins: A Practical Approach is a unique guide to the latest methods is use, written by the acknowledged experts in the field. Detailed protocols are provided for all the key techniques, and the relevant background material is included. This book is an essential manual for a wide range of scientists studying the modification of protein by lipids, including membrane and protein biochemists, cell biologists, immunologists, bacteriologists, parasitologists, and virologists.

The number of protein sequences grows each year, yet the number of structures deposited in the Protein Data Bank remains relatively small. The importance of protein structure prediction cannot be overemphasized, and this volume is a timely addition to the literature in this field. Protein Structure Prediction: Methods and Protocols is a departure from the normal Methods in Molecular Biology series format. By its very nature, protein structure prediction demands that there be a greater mix of theoretical and practical aspects than is normally seen in this series. This book is aimed at both the novice and the experienced researcher who wish for detailed inf- mation in the field of protein structure prediction; a major intention here is to include important information that is needed in the day-to-day work of a research scientist, important information that is not always decipherable in scientific literature. Protein Structure Prediction: Methods and Protocols covers the topic of protein structure prediction in an eclectic fashion, detailing aspects of pred- tion that range from sequence analysis (a starting point for many algorithms) to secondary and tertiary methods, on into the prediction of docked complexes (an essential point in order to fully understand biological function). As this volume progresses, the authors contribute their expert knowledge of protein structure prediction to many disciplines, such as the identification of motifs and domains, the comparative modeling of proteins, and ab initio approaches to protein loop, side chain, and protein prediction.

Electron Microscopy Methods and Protocols is designed for the established researcher as a manual for extending knowledge of the field. It is also for the newcomer who wishes to move into the field. A wide range of applications for the examination of cells, tissues, biological macromolecules, molecular structures, and their interactions are discussed. We have tried to gather together methods that we consider to be those most generally appli- ble to current research in both cell and molecular biology. Each chapter c- tains a set of related practical protocols with examples provided by experts who have first-hand knowledge of the techniques they describe. The individual chapters are grouped according to similarities in their specimen preparation and methodology. Methods are presented in detail, in a step-by-step fashion, using reproducible protocols the authors have personally checked. During the last decade, the scientific literature describing the use of colloidal gold as an immunocytochemical marker has increased at an ex- nential rate, and this trend is expected to continue. We have included a large number of variations on the immunogold labeling technique. In both the ne- tive staining and cryo chapters, authors emphasize the “immunological app- cations” in order to correlate as fully as possible with the emphasis on immunogold labeling in the other chapters. Electron Microscopy Methods and Protocols commences with the routine preparation of biological material for classical transmission electron microscopy involving tissue fixation, embedding, and sectioning (Chap. 1).