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• W2938998332 startingPage "1634" @default.
• W2938998332 abstract "In vitro reconstitutions of microtubule assemblies have provided essential mechanistic insights into the molecular bases of microtubule dynamics and their interactions with associated proteins. The tubulin code has emerged as a regulatory mechanism for microtubule functions, which suggests that tubulin isotypes and post-translational modifications (PTMs) play important roles in controlling microtubule functions. To investigate the tubulin code mechanism, it is essential to analyze different tubulin variants in vitro. Until now, this has been difficult, as most reconstitution experiments have used heavily post-translationally modified tubulin purified from brain tissue. Therefore, we developed a protocol that allows purification of tubulin with controlled PTMs from limited sources through cycles of polymerization and depolymerization. Although alternative protocols using affinity purification of tubulin also yield very pure tubulin, our protocol has the unique advantage of selecting for fully functional tubulin, as non-polymerizable tubulin is excluded in the successive polymerization cycles. It thus provides a novel procedure for obtaining tubulin with controlled PTMs for in vitro reconstitution experiments. We describe specific procedures for tubulin purification from adherent cells, cells grown in suspension cultures and single mouse brains. The protocol can be combined with drug treatment, transfection of cells before tubulin purification or enzymatic treatment during the purification process. The amplification of cells and their growth in spinner bottles takes ~13 d; the tubulin purification takes 6–7 h. The tubulin can be used in total internal reflection fluorescence (TIRF)-microscopy-based experiments or pelleting assays for the investigation of intrinsic properties of microtubules and their interactions with associated proteins. Tubulin with controlled posttranslational modifications can be purified by cycles of polymerization and depolymerization from cultured cells or single mouse brains for use in in vitro reconstitution assays." @default.
• W2938998332 created "2019-04-25" @default.
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• W2938998332 date "2019-04-17" @default.
• W2938998332 modified "2023-10-17" @default.
• W2938998332 title "Purification of tubulin with controlled post-translational modifications by polymerization–depolymerization cycles" @default.
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• W2938998332 doi "https://doi.org/10.1038/s41596-019-0153-7" @default.
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• W2938998332 hasPublicationYear "2019" @default.
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