Katsuki, Miho, Muto, Etsuko and Cross, Robert A.. (2011) Preparation of dual-color polarity-marked fluorescent microtubule seeds. Methods in Molecular Biology (Clifton, N.J.), Vol.777 . pp. 117-126. ISSN 1064-3745

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Abstract

Assaying microtubule dynamics in vitro requires stabilized nucleation centers, a method to immobilize individual microtubules onto a surface, and a specialized microscope to image the microtubule. Microtubules are polar structures with different dynamic properties at the plus and minus ends. However, the dynamics of the two ends can be modified by the addition of other proteins, such as microtubule plus-end-tracking proteins (+TIPs), so that it becomes impossible to distinguish the microtubule polarity by measuring the differences in the dynamic properties of the ends alone. In this chapter, we describe a method for labeling tubulin protein with N-hydroxysuccinimide ester fluorescent dyes, enabling the formation of dual-color polarity-marked stable microtubule seeds that can be immobilized onto a microscopic cover glass for imaging by fluorescence microscopy. These seeds create functional nucleation centers for the growth of dynamic microtubules.

Item Type:	Journal Article
Subjects:	R Medicine > R Medicine (General)
Divisions:	Faculty of Medicine > Warwick Medical School > Biomedical Cell Biology Faculty of Medicine > Warwick Medical School
Series Name:	Methods in Molecular Biology
Journal or Publication Title:	Methods in Molecular Biology (Clifton, N.J.)
Publisher:	Humana Press, Inc.
ISSN:	1064-3745
Book Title:	Microtubule Dynamics
Date:	31 August 2011
Volume:	Vol.777
Page Range:	pp. 117-126
Identification Number:	10.1007/978-1-61779-252-6_9
Status:	Peer Reviewed
Publication Status:	Published
URI:	http://wrap.warwick.ac.uk/id/eprint/46275

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