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Proteomic strategy.

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posted on 27.05.2011 by Mary Kate Bonner, Daniel S. Poole, Tao Xu, Ali Sarkeshik, John R. Yates III, Ahna R. Skop

CHO cells were synchronized at metaphase by successive thymidine and nocodazole blocks. Taxol was applied to stabilize the spindle structure. Cells were lysed in a hypotonic solution to release spindles. Four spindle samples were each trypsinized and MudPIT was performed. Proteins were identified by tandem liquid chromatography and tandem mass spectrometry (LC/LC-MS/MS), and the metaphase spindle proteome was assembled. Proteins in at least three mass spec runs appeared in the final list.

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