Methods In Molecular Biology Adhesion Protein Protocols

The second edition of Adhesion Protein Protocols combines traditional techniques with cutting-edge and novel techniques that can be adapted easily to different molecules and cell types. The topics discussed include novel techniques for studying cell-cell adhesion, neutrophil chemotaxis, in vitro assays used to study leukocyte migration through monolayers of cultured endothelial cells, and novel techniques to purify pseudopodia from migratory cells. The protocols discussed in this volume are suitable for both novice and expert scientists, who will gain further insight into the complex and incompletely understood processes involved in cellular adhesion.

This detailed book collects methodologies exploring mechanobiology, the involvement of mechanical forces in cell fate specification and in controlling single and collective cell behaviors such as directed migration, morphogenesis, wound healing, and the immune response. The volume features methods to quantify the mechanical properties of cells and adhesion proteins, to expose cells to external mechanical forces, to quantitatively characterize mechano-responses at various scales, to measure forces applied by cells on the extracellular matrix, as well as chapters on force measurement inside cells, probing cell signaling and gene expression in response to force, and biophysical modeling of cell shape and protein dynamics. Written for the highly successful Methods in Molecular Biology series, chapters include introductions to their respective topics, lists of the necessary material and reagents, step-by-step and readily reproducible protocols, and tips on troubleshooting and avoiding known pitfalls. Authoritative and practical, Mechanobiology: Methods and Protocols aims to provide meaningful tools for cell and developmental biologists approaching the study of cell and tissue dynamics from a mechanobiological perspective, molecular biologists interested in the effects of force on proteins, as well as for cancer biologists and biophysicists.

Integrins play pivotal roles not only across a wide range of physiological processes including tissue morphogenesis, immune responses, wound healing, and regulation of cell growth and differentiation, but also in numerous pathological phenomena such as autoimmunity, thrombosis, and cancer metastasis/progression. Therefore, investigations on integrins often demand multi-disciplinary approaches, making researchers long for a handy collection of comprehensive and practical protocols that detail experimental methods for studying integrin and related cell adhesion molecule functionality. Integrin and Cell Adhesion Molecules: Methods and Protocols aims to provide readers not only with basic protocols in studying integrin functions, but also with summaries on those state-of-the-art technologies that have been utilized for understanding integrin functionality at the cellular, molecular, structural, and organismal levels. Divided into six convenient sections, this detailed volume covers basic protocols for the study of integrin and related cell adhesion molecule functionality in vitro, illustrates structural biology approaches for studying integrins and related cell adhesion molecules, focuses on emerging imaging technologies for investigating cell migration, presents strategies to elucidate signaling through cell adhesion molecules, includes experimental techniques to investigate integrin functions at organismal levels in a physiological context, and showcases the most promising methods and technologies for the development of novel therapeutics and diagnostics. Written in the successful Methods in Molecular BiologyTM series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible protocols, and notes on troubleshooting and avoiding known pitfalls. Both experts and non-experts in the scientific community who wish to study cell adhesion molecules and diagnostics will find Integrin and Cell Adhesion Molecules: Methods and Protocols authoritative, easily accessible, and vastly informative.

The second edition of Adhesion Protein Protocols combines traditional techniques with cutting-edge and novel techniques that can be adapted easily to different molecules and cell types. The topics discussed include novel techniques for studying cell-cell adhesion, neutrophil chemotaxis, in vitro assays used to study leukocyte migration through monolayers of cultured endothelial cells, and novel techniques to purify pseudopodia from migratory cells. The protocols discussed in this volume are suitable for both novice and expert scientists, who will gain further insight into the complex and incompletely understood processes involved in cellular adhesion.

It is now widely accepted that much of the dynamic function of cells and tissues is regulated from outside the cell by the extracellular matrix. In ad- tion to its conventional role in providing a scaffold for building tissues, the extracellular matrix acts as a directional highway for cellular movement and provides instructional information for promoting survival, proliferation, and differentiation. Indeed, the extracellular matrix is beginning to take a starring role in the choreography of cell and tissue function. The diverse roles of the extracellular matrix are reflected in its highly complicated structure, consisting of an ever increasing number of components. Yet the mechanisms of extracellular matrix assembly and how they influences cell behavior are only just beginning to be understood. In order to solve these problems new methodologies are, of necessity, being developed. Many of these technologies are highly sophisticated and are currently available only in a ha- ful of laboratories. However, we believe that they can readily be transported and established by other researchers. Thus, the purpose of Extracellular Matrix Protocols is to present some of these complicated techniques in a style that is relatively easy to reproduce.

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.

In The Protein Protocols Handbook, I have attempted to provide a cross-section of analytical techniques commonly used for proteins and peptides, thus providing a benehtop manual and guide both for those who are new to the protein chemistry laboratory and for those more established workers who wish to use a technique for the first time. We each, of course, have our own favorite, commonly used gel system, g- staining method, blotting method, and so on; I'm sure you will find yours here. H- ever, I have also described a variety of altematives for many of these techniques; though they may not be superior to the methods you commonly use, they may nev- theless be more appropriate in a particular situation. Only by knowing the range of techniques that are available to you, and the strengths and limitations of these te- niques, will you be able to choose the method that best suits your purpose.