Kinesin Protocols

VERNOS, I ed.
cover image
Publication
Totowa, NJ : Humana Press, 2001.
ISBN
9781592590698
Printed edition: 9780896037663
Physical Description
XI, 258 p. online resource.
Series
Methods in Molecular Biology™ Vol 164
URL
Language
eng
 
 
 
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$a Purification of Kinesin from the Brain -- RT-PCR for the Identification of Developmentally Regulated Novel Members of the Kinesin-like Superfamily -- Expression Cloning with Pan Kinesin Antibodies -- Expression of Kinesin in Escherichia coli -- Plasmids for Expression of Chimeric and Truncated Kinesin Proteins -- Preparation of Recombinant Kinesin Superfamily Proteins Using the Baculovirus System -- Assays for Kinesin Microtubule-Stimulated AT Pase Activity -- An Improved Microscope for Bead and Surface-Based Motility Assays -- Use of Photonic Force Microscopy to Study Single-Motor-Molecule Mechanics -- Assays for Microtubule-Destabilizing Kinesins -- Green Fluorescent Protein as a Tag for Molecular Motor Proteins -- In Vitro Reconstitution of Endosome Motility Along Microtubules -- Approaches to Study Interactions Between Kinesin Motors and Membranes -- Microinjection Methods for Analyzing the Functions of Kinesins in Early Embryos -- The Use of Dominant Negative Mutants to Study the Function of Mitotic Motors in the In Vitro Spindle Assembly Assay in Xenopus Egg Extracts -- A Dominant Negative Approach for Functional Studies of the Kinesin II Complex -- Identification of Kinesin-Associated Proteins -- Assaying Spatial Organization of Microtubules by Kinesin Motors -- Crystallization of Kinesin -- Structural Analysis of the Microtubule–Kinesin Complex by Cryo-Electron Microscopy.
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$a It is now clear that kinesin-like-proteins (KLP), generally thought to be responsible for the transport of cellular cargoes, are involved in many different cellular processes now being widely investigated. In Kinesin Protocols, Isabelle Vernos and a panel of hands-on experts present their most productive and reproducible techniques for the identification, purification, and characterization of the kinesin superfamily of microtubule-dependent motors. The methods range from the most basic to the most sophisticated and include step-by-step instructions and extensive cautionary notes to ensure experimental success. Among the approaches discussed are methods to express and purify kinesins in different systems, to characterize microtubule-enhanced ATPase activity and motility properties, and to test microtubule destabilizing activity. Detailed examples of how to address functional studies are also presented, along with some very new methods for studying the role of KLP in the organization of microtubules in three dimensions. There are also advanced methods for the study of kinesins at the structural level. Comprehensive and highly practical, Kinesin Protocols makes available all the key basic and cutting-edge methods needed successfully to study the multifaceted world of kinesin-like proteins and to explore their many functions.
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Subject
LIFE SCIENCES
CELL BIOLOGY
METHODS AND PROTOCOLS
E-BOOKS
Summary
It is now clear that kinesin-like-proteins (KLP), generally thought to be responsible for the transport of cellular cargoes, are involved in many different cellular processes now being widely investigated. In Kinesin Protocols, Isabelle Vernos and a panel of hands-on experts present their most productive and reproducible techniques for the identification, purification, and characterization of the kinesin superfamily of microtubule-dependent motors. The methods range from the most basic to the most sophisticated and include step-by-step instructions and extensive cautionary notes to ensure experimental success. Among the approaches discussed are methods to express and purify kinesins in different systems, to characterize microtubule-enhanced ATPase activity and motility properties, and to test microtubule destabilizing activity. Detailed examples of how to address functional studies are also presented, along with some very new methods for studying the role of KLP in the organization of microtubules in three dimensions. There are also advanced methods for the study of kinesins at the structural level. Comprehensive and highly practical, Kinesin Protocols makes available all the key basic and cutting-edge methods needed successfully to study the multifaceted world of kinesin-like proteins and to explore their many functions.
Table of Contents
Purification of Kinesin from the Brain -- RT-PCR for the Identification of Developmentally Regulated Novel Members of the Kinesin-like Superfamily -- Expression Cloning with Pan Kinesin Antibodies -- Expression of Kinesin in Escherichia coli -- Plasmids for Expression of Chimeric and Truncated Kinesin Proteins -- Preparation of Recombinant Kinesin Superfamily Proteins Using the Baculovirus System -- Assays for Kinesin Microtubule-Stimulated AT Pase Activity -- An Improved Microscope for Bead and Surface-Based Motility Assays -- Use of Photonic Force Microscopy to Study Single-Motor-Molecule Mechanics -- Assays for Microtubule-Destabilizing Kinesins -- Green Fluorescent Protein as a Tag for Molecular Motor Proteins -- In Vitro Reconstitution of Endosome Motility Along Microtubules -- Approaches to Study Interactions Between Kinesin Motors and Membranes -- Microinjection Methods for Analyzing the Functions of Kinesins in Early Embryos -- The Use of Dominant Negative Mutants to Study the Function of Mitotic Motors in the In Vitro Spindle Assembly Assay in Xenopus Egg Extracts -- A Dominant Negative Approach for Functional Studies of the Kinesin II Complex -- Identification of Kinesin-Associated Proteins -- Assaying Spatial Organization of Microtubules by Kinesin Motors -- Crystallization of Kinesin -- Structural Analysis of the Microtubule–Kinesin Complex by Cryo-Electron Microscopy.
Notes
Springer Protocols
Methods in Molecular Biology™, 1064-3745 ; 164