Purification of Fluorescently Labeled Saccharomyces cerevisiae Spindle Pole Bodies
| Autor: | Kimberly K. Fong, Trisha N. Davis, Beth Graczyk |
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| Rok vydání: | 2016 |
| Předmět: |
Centrosome
0301 basic medicine Tandem affinity purification Saccharomyces cerevisiae Proteins Staining and Labeling biology Saccharomyces cerevisiae Mitosis biology.organism_classification Mass Spectrometry Article Spindle pole body Cell biology Spindle apparatus Chromosome segregation 03 medical and health sciences 030104 developmental biology 0302 clinical medicine Spindle Pole Bodies Microtubule 030217 neurology & neurosurgery |
| Zdroj: | Methods in Molecular Biology ISBN: 9781493935406 |
| DOI: | 10.1007/978-1-4939-3542-0_12 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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