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Methods Mol Biol. 2016;1413:189-195. doi: 10.1007/978-1-4939-3542-0_12.

Purification of Fluorescently Labeled Saccharomyces cerevisiae Spindle Pole Bodies.

Author information

1
Department of Biochemistry, University of Washington, Seattle, WA 98195.
#
Contributed equally

Abstract

Centrosomes are components of the mitotic spindle responsible for organizing microtubules and establishing a bipolar spindle for accurate chromosome segregation. In budding yeast, Saccharomyces cerevisiae, the centrosome is called the spindle pole body, a highly organized trilaminar structure embedded in the nuclear envelope. Here we describe a detailed protocol for the purification of fluorescently labeled spindle pole bodes from S. cerevisiae. Spindle pole bodies are purified from yeast using a TAP-tag purification followed by velocity sedimentation.This highly reproducible TAP-tag purification method improves upon previous techniques and expands the scope of in vitro characterization of yeast spindle pole bodies. The genetic flexibility of this technique allows for the study of spindle pole body mutants as well as the study of spindle pole bodies during different stages of the cell cycle. The ease and reproducibility of the technique make it possible to study spindle pole bodies using a variety of biochemical, biophysical, and microscopic techniques.

KEYWORDS:

Centrosome; Mitosis; Purification; Spindle pole body (SPB); TAP-tag

PMID:
27193850
PMCID:
PMC4902105
DOI:
10.1007/978-1-4939-3542-0_12
[Indexed for MEDLINE]
Free PMC Article

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