A method for in situ mitotic spindle binding assay.
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.
Giet
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R
CNRS UPR41, Groupe Cycle Cellulaire, Université de Rennes I, Campus de Beaulieu, Rennes cedex, 35042, France.
Prigent
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C
D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis; D000918:Antibody Specificity; D064127:Aurora Kinases; D002417:Cattle; D018797:Cell Cycle Proteins; D002460:Cell Line; D004789:Enzyme Activation / Q000235:genetics; D019084:Fluorescent Antibody Technique, Indirect; D008870:Microtubules / Q000378:metabolism; D011485:Protein Binding / Q000187:drug effects; D011494:Protein Kinases / Q000276:immunology / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases; D011994:Recombinant Proteins / Q000378:metabolism; D008941:Spindle Apparatus / Q000737:chemistry* / Q000276:immunology / Q000378:metabolism*; D014981:Xenopus; D029867:Xenopus Proteins
D000911:Antibodies, Monoclonal; D018797:Cell Cycle Proteins; D011994:Recombinant Proteins; D029867:Xenopus Proteins; D011494:Protein Kinases; C112134:AURKA protein, Xenopus; D064127:Aurora Kinases; D017346:Protein Serine-Threonine Kinases