Protein Targeting Protocols

ISBN-13: 9780896034501 / Angielski / Miękka / 1998 / 331 str.

Roger A. Clegg

Opis

It is by no means a revelation that proteins are not uniformly distributed throughout the cell. As a result, the idea that protein molecules, because of the specificity with which they can engage in interactions with other proteins, may be aimed via these interactions at a restricted target, is a fundamental one in contemporary molecular life sciences. The target may be variously c- ceived as a specific molecule, a group of molecules, a structure, or a more generic type of intracellular environment. Because the concept of protein targeting is intuitive rather than expl- itly defined, it has been variously used by different groups of researchers in cell biology, biochemistry, and molecular biology. For those working in the field of intracellular signaling, an influential introduction to the topic was the seminal article by Hubbard & Cohen (TIBS 1993] 18, 172 177), which was based on the work of Cohen s laboratory on protein phosphatases. Sub- quently, the ideas that they discussed have been further developed and extended by many workers to other key intermediaries in intracellular sign- ing, including protein kinases and a great variety of modulator and adaptor proteins."

Recenzje

"... a valuable resource to the molecular biology community. The detailed protocols will greatly aid researchers..."-The Quarterly Review of Biology

Spis treści

Surface Plasmon Resonance: Measuring Protein Interactions in Real Time, G. Panayotou. Microcalorimetry of Protein-Protein Interactions, A. Cooper. Partitioning of Proteins in Triton X-114, J. G. Pryde. Co-immunoprecipitation: Identification of Interacting Proteins, N. G. Anderson. Overlay, Ligand Blotting, and Band-Shift Techniques to Study Kinase Anchoring, Z. E. Hausken, V. M. Coghlan, and J. D. Scott. Size-Exclusion Chromatography: Identification of Interacting Proteins, J. Beattie. Overlay and Bead Assay: Determination of Calcium Channel Subunit Interaction Domains, V. E. S. Scott, C. A. Gurnett, and K. P. Campbell. Use of GST-Fusion and Related Constructs for the Identification of Interacting Proteins, M. Harris. Molecular Genetic Approaches I: Two Hybrid Systems, G. B. Bolger. Molecular Genetic Approaches II: Expression-Interaction Cloning, M. Csukai and D. Mochly-Rosen. Molecular Genetic Approaches III: Determination of Protein Sequence Motifs Involved in Protein Targeting by Use of Coupled Transcription-Translation Systems, G. Scotland and M. D. Houslay. Molecular Genetic Approaches IV: Recombinant Expression of Wild-Type and Acylation-Resistant G-Protein a-Subunits Using Transient Transfection Systems, M. A. Grassie and G. Milligan. Use of Synthetic Peptides in the Dissection of Protein-Targeting Interactions, J. D. Scott and M. C. Faux. SH2 and SH3 Domains: Unraveling Signaling Networks with Peptide Antagonists, R. Stein. PH Domain of Serine-Threonine Protein Kinase B (RAC-PKB): Expression and Binding Assay for Phosphoinositides and Inositol Phosphates, M. Frech and B. A. Hemmings. Membrane Targeting via Protein N-Myristoylation, R. A. J. McIlhinney. Membrane Targeting via Protein Palmitoylation, M. Veit and M. F. G. Schmidt. Agonist-Mediated Turnover of G-Protein a-Subunit Palmitoyl Groups: Role in Membrane Insertion, M. A. Grassie and G. Milligan. Membrane Targeting via Protein Prenylation, J. H. Overmeyer, R. A. Erdman, and W. A. Maltese. Nuclear Protein Import in a Permeabilized Cell Assay, F. Melchior. Clathrin-Coated Vesicles and Targeting: Preparation of Adaptor Proteins, A. Pauloin. Analysis of the Sorting of Secretory Proteins to the Regulated Secretory Pathway: A Subcellular Fractionation Approach, E. Chanat, A. S. Dittié, and S. A. Tooze. Index.

Blurb

In Protein Targeting Protocols, master experimentalists describe, largely from the perspective of intracellular signaling, their best time-tested methods for detecting and analyzing the proteins and protein motifs involved in cellular targeting interactions. The techniques range from basic methods that reveal the existence of targeting interactions and characterize physicochemical properties to those for analyzing the more structurally elaborate systems involved in the movement of molecules between cellular compartments, with examples taken from nuclear transport, clathrin-coated vesicle traffic, and secretory sorting. The themes of targeting domains, particularly the SH-2, SH-3, and PH domains, and the principal targeting of lipid modifications-myristoylation, reversible palmitoylation, prenylation-are fully explored.

Protein Targeting Protocols offers a valuable collection of both basic and state-of-the-art techniques suitable for use in all areas of molecular cell biology by researchers interested in defining and characterizing the wide variety of protein interactions underlying targeting mechanisms. Readers will find it serves well as a firm methodological foundation for laboratories newly entering this field and as an indispensable reference work for those already well-established in it.