Mitosis

by McAinsh, Andrew D., Ph.D.

• ISBN13: 9781603279925
• ISBN10: 160327992X
• Edition: 1st
• Format: Hardcover
• Copyright: 2009-05-01
• Publisher: Humana Pr Inc

Summary

With the advent of modern methodology, the phenomenal complexity of the protein components and regulatory steps involved in mitosis has become approachable. In Mitosis: Methods and Protocols, experts in the field provide an up-to-date collection of methods and approaches that are used to investigate the mechanism of mitosis at the molecular level. Ideal for both experienced and novice scientists, the volume includes step-by-step methods covering inactivation of genes of interest, depletion of proteins of interest, biochemical and microscope-based techniques, and procedures to monitor and measure key mitotic processes. Written in the highly successful Methods in Molecular Biology'„¢ series format, chapters include brief introductions to the respective topics, lists of the necessary materials and reagents, readily reproducible laboratory protocols, and notes on troubleshooting and avoiding known pitfalls.Comprehensive and authoritative, Mitosis: Methods and Protocols combines imaging, biochemical, and genetic methodologies in order to best guide researchers in their attempts to better understand the crucial processes of mitotic regulation.

Table of Contents

Preface	p. v
Contributors	p. ix
Knock-in and Knock-out: The Use of Reverse Genetics in Somatic Cells to Dissect Mitotic Pathways	p. 1
Functional Dissection of Mitotic Regulators Through Gene Targeting in Human Somatic Cells	p. 21
RNAi in Drosophila S2 Cells as a Tool for Studying Cell Cycle Progression	p. 39
Production of Mitotic Regulators Using an Autoselection System for Protein Expression in Budding Yeast	p. 63
Hydrodynamic Analysis of Human Kinetochore Complexes During Mitosis	p. 81
Isolation of Protein Complexes Involved in Mitosis and Cytokinesis from Drosophila Cultured Cells	p. 99
Automated Live Microscopy to Study Mitotic Gene Function in Fluorescent Reporter Cell Lines	p. 113
Electron Tomography of Microtubule End-Morphologies in C. elegans Embryos	p. 135
Dissecting Mitosis with Laser Microsurgery and RNAi in Drosophila Cells	p. 145
Fluorescence Imaging of the Centrosome Cycle in Mammalian Cells	p. 165
Visualization of Fluorescence-Tagged Proteins in Fission Yeast: The Analysis of Mitotic Spindle Dynamics Using GFP-Tubulin Under the Native Promoter	p. 185
Analysing Kinetochore Function in Human Cells: Spindle Checkpoint and Chromosome Congression	p. 205
Probing Kinetochore Structure and Function Using Xenopus laevis Frog Egg Extracts	p. 221
Live Cell Imaging of Kinetochore Capture by Microtubules in Budding Yeast	p. 233
The Spindle Checkpoint: Assays for the Analysis of Spindle Checkpoint Arrest and Recovery	p. 243
Measuring Proteolysis in Mitosis	p. 259
An In Vitro Assay for Cdc20-Dependent Mitotic Anaphase-Promoting Complex Activity from Budding Yeast	p. 271
In Vitro Assays for the Anaphase-Promoting Complex/Cyclosome (APC/C) in Xenopus Egg Extracts	p. 287
Preparation of Synchronized Human Cell Extracts to Study Ubiquitination and Degradation	p. 301
Biochemical Analysis of the Anaphase Promoting Complex: Activities of E2 Enzymes and Substrate Competitive (Pseudosubstrate) Inhibitors	p. 313
Index	p. 331
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