Analysis of microtubule polymerization in vitro and during the cell cycle in Xenopus egg extracts.
Microtubules are dynamic polymers that participate in multiple cellular processes such as vesicular transport and cell division. Microtubule dynamics alter dramatically during the cell cycle. An excellent system to study microtubule dynamics is Xenopus egg extracts since it is a system that is open to manipulation. The extracts can be cycled between mitosis and interphase allowing the study of microtubules in these phases as well as during cell cycle transitions. Here, we provide simple assays to study microtubules in extracts and in vitro using purified components. Protocols are provided for the purification of frog tubulin, microtubule pelleting from extracts and in vitro, assembly of microtubule structures in extracts, and isolation of microtubule-associated proteins from extract. These methods can be used to analyze the effect of a protein of interest on the microtubule cytoskeleton.
Budde
Priya Prakash
PP
Howard Hughes Medical Institute, Department of Microbiology and Molecular Biology, Tufts University School of Medicine, MA 02111, USA.
Desai
Arshad
A
Heald
Rebecca
R
D000818:Animals; D002453:Cell Cycle / Q000502:physiology*; D002457:Cell Extracts; D002458:Cell Fractionation / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D008297:Male; D008869:Microtubule-Associated Proteins / Q000302:isolation & purification; D008870:Microtubules / Q000737:chemistry* / Q000378:metabolism / Q000648:ultrastructure*; D010063:Ovum / Q000378:metabolism / Q000648:ultrastructure; D014404:Tubulin / Q000302:isolation & purification; D014981:Xenopus
D002457:Cell Extracts; D008869:Microtubule-Associated Proteins; D014404:Tubulin