Protein Targeting Protocols

Expert researchers describe the key methods for detecting and analyzing proteins and protein motifs involved in targeting interactions, mostly from the perspective of intracellular signaling. The techniques include those designed to reveal the existence of targeting interactions and characterize their physico-chemical properties, to identify and isolate interacting proteins on the basis of the 'bait-prey' principle, to target domains, and to modify lipids. The methods are richly detailed for use by those researchers interested in defining and characterizing the protein-protein interactions that underlie targeting mechanisms in all areas of molecular cell biology. Surface Plasmon Resonance : Measuring Protein Interactions In Real Time / George Panayotou -- Microcalorimetry Of Protein-protein Interactions / Alan Cooper -- Partitioning Of Proteins In Triton X-114 / James G. Pryde -- Co-immunoprecipitation : Identification Of Interacting Proteins / Neil G. Anderson -- Overlay, Ligand Blotting, And Band-shift Techniques To Study Kinase Anchoring / Zachary E. Hausken, Vincent M. Coghlan, John D. Scott -- Size-exclusion Chromatography : Identification Of Interacting Proteins / James Beattie -- Overlay And Bead Assay : Determination Of Calcium Channel Subunit Interaction Domains / Victoria E. Scott, Christina A. Gurnett, Kevin P. Campbell -- Use Of Gst-fusion And Related Constructs For The Identification Of Interacting Proteins / Mark Harris -- Molecular Genetic Approaches. 1, Two-hybrid Systems / Graeme B. Bolger -- Molecular Genetic Approaches. 2, Expression-interaction Cloning / Michael Csukai, Daria Mochly-rosen --^ Molecular Genetic Approaches. 3, Determination Of Protein Sequence Motifs Involved In Protein Targeting By Use Of Coupled Transcription-translation Systems / Grant Scotland, Miles D. Houslay -- Molecular Genetic Approaches. 4, Recombinant Expression Of Wild-type And Acylation-resistant G-protein [alpha]-subunits Using Transient Transfection Systems / Morag A. Grassie, Graeme Milligan -- Use Of Synthetic Peptides In The Dissection Of Protein-targeting Interactions / John D. Scott, Maree C. Faux -- Sh2 And Sh3 Domains : Unraveling Signaling Networks With Peptide Antagonists / Rob Stein -- Ph Domain Of Serine-threonine Protein Kinase B (rac-pkb) : Expression And Binding Assay For Phosphoinositides And Inositol Phosphates / Matthias Frech, Brian A. Hemmings -- Membrane Targeting Via Protein N-myristoylation / R.a. Mcilhinney -- Membrane Targeting Via Protein Palmitoylation / Michael Veit, Michael F. Schmidt --^ Agonist-mediated Turnover Of G-protein [alpha]-subunit Palmitoyl Groups : Role In Membrane Insertion / Morag A. Grassie, Graeme Milligan -- Membrane Targeting Via Protein Prenylation / Jean H. Overmeyer, Robert A. Erdman, William A. Maltese -- Nuclear Protein Import In A Permeabilized Cell Assay / Frauke Melchior -- Clathrin-coated Vesicles And Targeting : Preparation Of Adaptor Proteins / Alain Pauloin -- Analysis Of The Sorting Of Secretory Proteins To The Regulated Secretory Pathway : A Subcellular Fractionation Approach / Eric Chanat, Andrea S. Dittié, Sharon A. Tooze. Edited By Roger A. Clegg. Includes Index. Includes Bibliographical References And Index. 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. Master experimentalists describe, largely from the perspective of intracellular signaling, their best methods for detecting and analyzing the proteins and protein motifs involved in 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. This book will serve 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 Describes from the perspective of intracellular signaling the best methods for detecting and analyzing the proteins and protein motifs involved in targeting interactions. This book includes techniques ranging from basic methods that reveal the existence of targeting interactions to those for analyzing the more structurally elaborate systems. 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.