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A method for in situ mitotic spindle binding assay.

Abstract : The Xenopus centrosome protein kinase pEg2, involved in spindle assembly, binds to microtubules polymerized in vitro. We have developed a method to investigate the affinity of purified recombinant pEg2 protein for the cellular mitotic spindle. Briefly, cells grown on coverslips are fixed, permeabilized, and incubated with recombinant pEg2 protein. Localization of the protein is revealed by probing with a specific monoclonal antibody that recognizes recombinant but not endogenous pEg2. Using this method we show that recombinant pEg2 binds to microtubules in vitro, while, in vivo, pEg2 localized only to the mitotic spindle and not the interphase microtubule network. We also demonstrate that the catalytic activity of pEg2 is not necessary for its binding ability. This technique can be used to analyze the binding of various tagged proteins to cellular mitotic spindle.
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https://www.hal.inserm.fr/inserm-00966040
Contributor : Claude Prigent Connect in order to contact the contributor
Submitted on : Wednesday, March 26, 2014 - 9:01:23 AM
Last modification on : Thursday, January 14, 2021 - 11:15:27 AM

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Régis Giet, Claude Prigent. A method for in situ mitotic spindle binding assay.. Experimental Cell Research, Elsevier, 1998, 244 (2), pp.470-3. ⟨10.1006/excr.1998.4218⟩. ⟨inserm-00966040⟩

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